Antigen presenting cell assay

ABSTRACT

Disclosed herein are methods for diagnosing or predicting acute cellular and/or humoral rejection in a subject. In one example, a method of assessing organ rejection includes contacting a first sample comprising antigen presenting cells (APCs) obtained from a subject in need of or having received an organ transplant with a donor antigen from a donor under conditions sufficient to induce uptake of the donor antigen; contacting a second sample comprising APCs obtained from the subject in need of or having received an organ transplant with a third-party antigen under conditions sufficient to induce uptake of the third-party antigen; and determining an antigen presenting index by determining a ratio of uptake of the donor antigen in the first sample to uptake of the third-party antigen in the second sample, wherein the ratio of greater than one indicates organ rejection and the APCs are monocytes or monocyte-derived cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/052,391, filed on Oct. 11, 2013, which is a Continuation-In-Part ofU.S. patent application Ser. No. 13/639,428, filed Oct. 4, 2012, nowU.S. Pat. No. 9,322,827, which is the U.S. National Stage ofInternational Application No. PCT/US2011/031705, filed Apr. 8, 2011,which was published in English under PCT Article 21(2), which in turnclaims the benefit of U.S. Provisional Application No. 61/322,234, filedApr. 8, 2010, all of which applications are hereby incorporated byreference in their entirety. U.S. patent application Ser. No. 14/052,391claims the benefit of U.S. Provisional Patent Application No.61/713,419, filed Oct. 12, 2012, which is hereby incorporated byreference in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. AI073895awarded by the National Institutes of Health. The government has certainrights in this invention.

FIELD OF THE DISCLOSURE

This disclosure relates to the field of immunology and in particular, tomethods for diagnosing and/or predicting transplant rejection, such asthe acute cellular rejection or antibody-mediated (humoral) rejection ofan organ transplant or graft-versus-host disease.

BACKGROUND

Transplanted organs and tissues are vulnerable to rejection; acutecellular rejection (ACR) and humoral rejection (HR) are two forms oftransplant rejection. Either process begins with uptake of a foreignantigen by an antigen presenting cell (APC). An APC presents the antigento effector cells, such as T- and B-lymphocytes. The way in which APCspresent the antigen to the effector cells determines whether the immunesystem reacts with an inflammatory response, or is tolerized to theantigen. The inflammatory response to a transplanted organ is calledalloresponse. An effector T-cell inflammatory alloresponse mediates ACR,while an effector B-cell inflammatory alloresponse includes maturationto antibody secreting memory B-cells (also known as plasma cells) whichmediate HR.

In order to identify patients at high risk for rejecting a transplant,cardiac/organ transplantation candidates are prospectively tested foranti-HLA antibodies against lymphocytes from a panel of subjectsrepresentative of the major HLA allotypes, collectively referred to asmeasurements of panel-reactive antibodies (PRA). In addition topredicting an increased likelihood of donor-specific anti-HLA antibodiesand a consequent risk of early graft failure related to humoralrejection, several studies have shown that high levels of pretransplantPRA in allograft recipients are associated with adverse post-transplantoutcome when compared to patients with low or negative reactivity. HighPRA levels have been associated, in some studies, with increasedfrequency of acute cellular rejection, decreased long-term graftsurvival, and increased mortality. Moreover, the onset of acceleratedcoronary artery disease (CAD) in cardiac transplant recipients, themajor limitation to long-term graft survival, has been associated withthe presence of anti-HLA antibodies. Since accelerated CAD in thesepatients may be a consequence of cumulative episodes of high-gradecellular rejections, it is possible that this association may actuallyreflect a relationship between anti-HLA antibodies and acute cellularrejection. However, PRA, and other test are not highly sensitive orspecific. Thus, a need remains for a sensitive and specific assay foracute cellular and humoral rejection.

SUMMARY OF THE DISCLOSURE

The B-cell arises from bone marrow progenitor cells, which progressthrough multiple stages such as the pro-, pre-, and transitional stagesinto the naive B-cell. The B-cell has many functions. It can serve as anantigen presenting cell, which activates T-cytotoxic cells towardeffector function, can activate naive or memory Th1 cells, or evolvesinto long-lived memory cell and transforms into an antibody secretingplasma cell with T-cell help, and perpetuates antibody responses toautoantigens. Antigen is sensed by the B-cell via the B-cell receptor,or the immunoglobulin molecule.

Antigen recognition, uptake and presentation are early steps in the manyfunctions fulfilled by the B-cell. The inventors have developed methodsand assays to measure this antigen presenting function for the earlydetection of activity or severity for a variety of immunologicaldiseases caused by foreign and other antigens. These antigens includebut are not limited to transplanted organs and tissues and cells,infectious pathogens, allergens, autoantigens (antigens arising fromself, which would normally not evoke an immune response) and tumorantigens.

As such, disclosed herein are methods and assays for the diagnosis andprediction of B-cell activity, such as acute cellular or humoralrejection. Monitoring antigen uptake and presentation by APCs is used todetermine whether the effector cell response will result in ACR, HR, oran absence thereof. In several examples, the disclosed assay is used todiagnose or predict organ transplant rejection, graft-versus-hostdisease (GVHD) or immunity to an antigen. For example, the assay is usedto diagnose or predict GVHD after solid organ, bone-marrow, stem celltransplantation or a combination thereof. In other examples, the assayis used to predict immunity to tumor antigens, autoantigens, pathogenantigens or a combination thereof.

Also provided is a method of assessing organ rejection includingcontacting a first sample that includes APCs obtained from a subject inneed of or having received an organ transplant from a donor, with adonor antigen from the donor, under conditions sufficient to induceuptake of the donor antigen, and measuring the uptake of the donorantigen. The method also includes contacting a second sample thatincludes APCs obtained from the subject in need of or having received anorgan transplant, with a third-party antigen under conditions sufficientto induce uptake of the third-party antigen, and measuring the uptake ofthe third party antigen. The ratio of uptake of the donor antigen in thefirst sample to uptake of the third-party antigen in the second sampleis then determined. If donor antigen uptake exceeds third-party antigenuptake in the form a ratio greater than one than the subject has, or islikely to develop, organ rejection. In some embodiments, the APCpreparation consists of peripheral blood leukocytes. In some particularembodiments, the APC preparation consists of B-cells.

Also disclosed herein is a method for assessing B-cell rejection in asubject by determining an antigen presenting index (API). The methodincludes comparing uptake by recipient APCs of a donor antigen to theuptake by recipient APCs of a third-party antigen. An API of greaterthan one predicts with a sensitivity of at least 90% and a specificityof at least 90% for an increased risk of acute cellular or humoralrejection, or the presence of acute cellular or humoral rejection.

Also provided is method of assessing GVHD in a subject. The methodincludes assessing GVHD by contacting a first sample that includes donorAPCs obtained from a sample of donor bone marrow or stem cells beforetransplantation, or from the recipient who has received donor bonemarrow or stem cells, with a recipient antigen from a subject havingreceived a bone marrow transplant or a stem cell transplant therecipient, under conditions sufficient to induce uptake of the recipientantigen, and measuring the uptake of the recipient antigen. The methodalso includes contacting a second sample that includes donor APCsobtained from a sample of donor bone marrow or stem cells beforetransplantation, from the recipient who has received donor bone marrowor stem cells, with a third-party antigen under conditions sufficient toinduce uptake of the third-party antigen, and measuring the uptake ofthe third party antigen. The ratio of uptake of the recipient antigen inthe first sample to uptake of the third-party antigen in the secondsample is then determined. A ratio of greater than one indicates thatthe subject has, or is likely to develop, GVHD. In some embodiments, theAPC preparation consists of peripheral blood leukocytes. In someexamples, the APC preparation consists of a B-cell, such as a donorB-cell.

Further provided are methods for determining the B-cell response toother antigens, including antigens from allergens, infectious pathogens,tumors or associated with autoimmune disorders/diseases. Infectiouspathogens include bacteria, fungi, protists, prions and/or viruses.These additional uses can be either alone or in addition to diagnosingor predicting organ transplant rejection.

Also provided are methods of assessing organ rejection by utilizingadditional types of antigen presenting cells, such as monocytes ormonocyte-derived cells (e.g., monocyte-derived dendritic cells). In someexamples, a method of assessing organ rejection, comprises contacting afirst sample comprising antigen presenting cells (APCs) obtained from asubject in need of or having received an organ transplant with a donorantigen from a donor under conditions sufficient to induce uptake of thedonor antigen; contacting a second sample comprising APCs obtained fromthe subject in need of or having received an organ transplant with athird-party antigen under conditions sufficient to induce uptake of thethird-party antigen; and determining an antigen presenting index bydetermining a ratio of uptake of the donor antigen in the first sampleto uptake of the third-party antigen in the second sample, wherein theratio of greater than one indicates organ rejection and the APCs aremonocytes or monocyte-derived cells.

Methods for assessing acute cellular and/or humoral rejection in asubject by using additional types of antigen presenting cells, such asmonocytes or monocyte-derived dendritic cells are disclosed. In someexamples, a method include for assessing acute cellular and/or humoralrejection comprises determining an antigen presenting index by (i)contacting a first portion of a biological sample comprising antigenpresenting cells (APCs) obtained from a subject in need of or havingreceived an organ transplant from a donor with a donor antigen from adonor under conditions sufficient to induce uptake of the donor antigen;(ii) contacting a second portion of the biological sample comprisingAPCs obtained from the subject in need of or having received an organtransplant with a third-party antigen under conditions sufficient toinduce uptake of the third-party antigen; and (iii) determining theratio of uptake of the donor antigen in the first portion of thebiological sample to uptake of the third-party antigen in the secondportion of the biological sample, wherein the APCS are monocytes ormonocyte-derived cells and the antigen presenting index of greater thanone predicts with a sensitivity of at least 90%, such as at least 95%and a specificity of at least 75% for an increased risk of acutecellular rejection and/or humoral rejection.

The foregoing and other features of the disclosure will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a series of scatterplots of flow cytometry data illustratingantigen uptake in rejectors versus non-rejectors. In the rejector (upperpanels), 23.6% recipient B-cells present donor antigen (middle upperpanel), compared with 4.8% recipient B-cells which present third-partyantigen (right upper panel) for an API of 4.91. In the non-rejector(lower panels), 35.9% of recipient B-cells present third-party antigen(lower right panel), but only 13.3% present donor antigen (lower middlepanel). The API is 0.37 in this non-rejector.

FIG. 2 is a graph of the API from 28 children with liver or intestinetransplantation at three different time points. The majority ofnon-rejectors (bottom panel) remained below the rejection-risk thresholdAPI of <1.11 at all three timepoints. The majority of rejectors (blacklines, top panel) were at or above the rejection-risk threshold IR of1.11 before and during the first 1-60-days after transplantation, butbelow this threshold during the 61-200-day period after transplantation.Error bars represent the standard error of the mean.

FIG. 3 is a set of graphs of the API of CD19+B-cells obtained using“actual” donor and “surrogate” donor peripheral blood leukocytes (PBL)plotted for 7 children with liver or intestine transplantation.Surrogate donor consists of peripheral blood leukocytes from healthynormal human subjects which are HLA-matched to the actual donor, andtherefore resemble this actual donor Correlation between API obtainedwith either stimulator were highly significant for either recipientpopulation (left panel). Assignment of rejector (R) or non-rejector (NR)status based on a rejection-risk threshold API of ≥1.115 was the samewith either stimulator for either recipient population (right panel).

FIG. 4 is a bar graph illustrating the cross-sectional cohort API ofCD14+ monocytes.

FIG. 5 is a graph illustrating longitudinal changes in API of CD14+monocytes.

FIG. 6 is a bar graph illustrating combined CD14+ monocyte API resultsfrom longitudinal and cross-sectional cohorts.

FIG. 7 is a scatter plot illustrating the correlation between antigenpresenting indices of the B-cell and monocyte in 35 samples.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS I. Abbreviations

ACR: acute cellular rejection

APC: antigen presenting cell

API: antigen presenting index

CD: cluster of differentiation

HR: humoral rejection

LTx: liver transplantation

MLR: mixed lymphocyte response

OVA: ovalbumin

PBL: peripheral blood leukocytes

SBTx: small bowel transplantation

II. Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular forms“a,” “an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. For example, the term “comprising a nucleicacid molecule” includes single or plural nucleic acid molecules and isconsidered equivalent to the phrase “comprising at least one nucleicacid molecule.” The term “or” refers to a single element of statedalternative elements or a combination of two or more elements, unlessthe context clearly indicates otherwise. As used herein, “comprises”means “includes.” Thus, “comprising A or B,” means “including A, B, or Aand B,” without excluding additional elements.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting.

Allograft: A transplant of an organ, tissue, bodily fluid or cell fromone individual to a genetically nonidentical individual of the samespecies. As used herein, “allogeneic” encompasses a geneticallydifferent phenotype present in non-identical individuals of the samespecies. Allogeneic examples include blood group phenotypes andimmunoantigeneic allotypes. An “alloantigen” encompasses any antigenrecognized by different individuals of the same species. Organisms,cells, tissues, organs, and the like from, or derived from, a singleindividual, or from a genetically identical individual are “autologous.”“Transplant rejection” refers to a partial or complete immune responseto a transplanted cell, tissue, organ, or the like on or in a recipientof the transplant due to an immune response to an allogeneic graft.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: A polypeptide ligand that includes at least a light chain orheavy chain immunoglobulin variable region and specifically binds anepitope of an antigen. Antibodies can include monoclonal antibodies,polyclonal antibodies, or fragments of antibodies.

The term “specifically binds” refers to, with respect to an antigen, thepreferential association of an antibody or other ligand, in whole orpart, with a specific polypeptide. A specific binding agent bindssubstantially only to a defined target. It is recognized that a minordegree of non-specific interaction may occur between a molecule, such asa specific binding agent, and a non-target polypeptide. Nevertheless,specific binding can be distinguished as mediated through specificrecognition of the antigen. Although selectively reactive antibodiesbind antigen, they can do so with low affinity. Specific bindingtypically results in greater than 2-fold, such as greater than 5-fold,greater than 10-fold, or greater than 100-fold increase in amount ofbound antibody or other ligand (per unit time) to a target polypeptide,such as compared to a non-target polypeptide. A variety of immunoassayformats are appropriate for selecting antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow & Lane,Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork (1988), for a description of immunoassay formats and conditionsthat can be used to determine specific immunoreactivity.

Antibodies can be composed of a heavy and a light chain, each of whichhas a variable region, termed the variable heavy (VH) region and thevariable light (VL) region. Together, the VH region and the VL regionare responsible for binding the antigen recognized by the antibody. Thisincludes intact immunoglobulins and the variants and portions of themwell known in the art, such as Fab′ fragments, F(ab)′2 fragments, singlechain Fv proteins (“scFv”), and disulfide stabilized Fv proteins(“dsFv”). A scFv protein is a fusion protein in which a light chainvariable region of an immunoglobulin and a heavy chain variable regionof an immunoglobulin are bound by a linker, while in dsFvs, the chainshave been mutated to introduce a disulfide bond to stabilize theassociation of the chains. The term also includes recombinant forms suchas chimeric antibodies (for example, humanized murine antibodies),heteroconjugate antibodies (such as bispecific antibodies). See also,Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford,Ill.); Kuby, Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

A “monoclonal antibody” is an antibody produced by a single clone of Blymphocytes or by a cell into which the light and heavy chain genes of asingle antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. These fused cells and their progeny are termed“hybridomas.” Monoclonal antibodies include humanized monoclonalantibodies.

In some examples, antibodies are labeled, for example with a fluorescentmarker that can aid in their detection.

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term is usedinterchangeably with the term “immunogen.” The term “antigen” includesall related antigenic epitopes. An “antigenic polypeptide” is apolypeptide to which an immune response, such as a T cell response or anantibody response, can be stimulated. “Epitope” or “antigenicdeterminant” refers to a site on an antigen to which B and/or T cellsrespond. T cells can respond to the epitope when the epitope ispresented in conjunction with an MHC molecule. Epitopes can be formedboth from contiguous amino acids (linear) or noncontiguous amino acidsjuxtaposed by tertiary folding of an antigenic polypeptide(conformational). Epitopes formed from contiguous amino acids aretypically retained on exposure to denaturing solvents whereas epitopesformed by tertiary folding are typically lost on treatment withdenaturing solvents. Normally, a B-cell epitope will include at leastabout 5 amino acids but can be as small as 3-4 amino acids. A T-cellepitope, such as a CTL epitope, will include at least about 7-9 aminoacids, and a helper T-cell epitope at least about 12-20 amino acids.Normally, an epitope will include between about 5 and 15 amino acids,such as, 9, 10, 12 or 15 amino acids. The amino acids are in a uniquespatial conformation. In one particular example, the antigen is anantigen obtained from a subject who is a donor, such as of an organ orof bone marrow, to another genetically different individual, suchantigen is referred to as a donor antigen. In one example, the donorantigen includes antigens from lymphocytes, leukocytes, such asperipheral blood leukocytes (including monocytes or monocyte-derivedcells, such as dendritic cells) or a combination thereof. In someexamples, donor antigen includes lysed cell membranes from donorperipheral blood leukocytes, spleen cells, or bone marrow cells. In anexample, donor antigen can be provided from a subject that had similarHLA-A, HLA-B, or HLA-DR loci profile as the donor. In other examples,the antigen is a third-party antigen (also referred to as a referenceantigen). A third-party antigen is an antigen that was not obtained fromthe organ donor or organ recipient and has no similarity to therecipient or donor (as indicated by measuring HLA-A, HLA-B and HLA-DRloci). Exemplary third-party antigen samples include lymphocytes,leukocytes, such as peripheral blood leukocytes (including monocytes ormonocyte-derived cells, such as dendritic cells) or a combinationthereof. For example, third-party antigen samples include lysed cellmembranes from donor peripheral blood leukocytes (including monocytes ormonocyte-derived cells, such as dendritic cells), spleen cells, or bonemarrow cells. An autoantigen is an antigen that under normal conditionswould not be a target of the immune system. However, the normalimmunological tolerance for such an antigen is lost in a subjectsuffering from a specific autoimmune disease and stimulates theproduction of autoantibodies.

Antigen Presenting Cells (APCs): Highly specialized cells that canprocess antigens and display their peptide fragments on the cell surfacetogether with molecules required for lymphocyte activation. For example,an APC is a cell that can present antigen bound to MHC class I or classII molecules to T cells. APCs include, but are not limited to,monocytes, macrophages, dendritic cells, B cells, T cells and Langerhanscells. A T cell that can present antigen to other T cells (includingCD4+ and/or CD8+ T cells) is an antigen presenting T cell (T-APC).

Antigen Presenting Index (API): A measure of the uptake (either bindingor internalization) of donor antigen in APCs of a subject, expressed asa ratio with the uptake of third-party antigen in APCs of the subject.An API>1 indicates an increased risk of rejection or the presence ofrejection in the subject. An API<1 indicates a decreased risk ofrejection or the absence of rejection in a subject. The API normalizesdonor antigen uptake to the uptake of a reference (third-party) antigenfor each person. The API is unique for each individual, but comparablebetween individuals.

B-cell: One of the two major types of lymphocytes. B-cells arise frombone marrow progenitor cells, which progress through multiple stagessuch as the pro-, pre- and transitional stages into the naive B-cell.The antigen receptor on B lymphocytes is a cell-surface immunoglobulinmolecule. Upon activation by an antigen, B-cells differentiate intocells producing antibody of the same specificity as their initialreceptor. B cells are also APCs.

An “immature B cell” is a cell that can develop into a mature B cell.Generally, pro-B cells (that express, for example, CD10) undergoimmunoglobulin heavy chain rearrangement to become pro B pre B cells,and further undergo immunoglobulin light chain rearrangement to becomean immature B cells. Immature B cells include T1 and T2 B cells. Thus,one example of an immature B cell is a T1 B that is anAA41^(hi)CD23^(lo) cell. Another example of an immature B cell is a T2 Bthat is an AA41^(hi)CD23^(hi) cell. Thus, immature B cells include B220expressing cells wherein the light and the heavy chain immunoglobulingenes are rearranged, and that express AA41. Immature B cells candevelop into mature B cells, which can produce immunoglobulins (e.g.,IgA, IgG or IgM). Mature B cells express characteristic markers such asCD21 and CD23 (CD23^(hi)CD21^(hi) cells), but do not express AA41. Insome examples, a B cell is one that expresses CD179^(hi), CD24, CD38 ora combination thereof. B cells can be activated by agents such aslippopolysaccharide (LPS) or IL-4 and antibodies to IgM.

B-cells have many functions. For example, a B-cell can serve as an APC(which activates T-cytotoxic cells toward effector function), activatenaive or memory Th1 cells, or evolve into long-lived memory cell andtransform into an antibody secreting plasma cell with T-cell help, andperpetuate antibody responses to autoantigens. Antigen is sensed by theB-cell via the B-cell receptor, or the immunoglobulin molecule.

CD5: A B-cell marker, also referred to as “cluster of differentiation5.” CD5⁺ B-cells are a class of atypical, self-renewing B cells foundmainly in the peritoneal and pleural cavities in adults and which have afar less diverse receptor repertoire than conventional B cells.

CD14: A monocyte marker. CD14 positive (CD14+) cells are monocytes thatcan differentiate into a host of different cells, including dendriticcells. In some types of monocytes, CD14 is co-expressed with CD16.

CD10: A B-cell marker. CD10 is primarily expressed on early B-cells andB-cell blasts, as well as T-cell precursors and bone marrow stromalcells. CD10 is an antigen that is a cell surface marker used in thediagnosis of human acute lymphocytic leukemia (ALL). CD10 is also knownas membrane metallo-endopeptidase (MME) and CALLA.

CD27: A protein expressed on medullary thymocytes, T-cells, naturalkiller (NK) cells and some B-cells. CD27 is a member of the TNF-receptorsuperfamily. This receptor is involved in the generation and long-termmaintenance of T cell immunity. It binds to ligand CD70, and plays arole in regulating B-cell activation and immunoglobulin synthesis. CD27transduces signals that lead to the activation of NF-κB and MAPK8/JNK.

CD154: A protein expressed on T-cells. CD154 is also known as CD40ligand. CD154 regulates B-cell function by engaging CD40 on the B-cellsurface. A defect in this gene results in an inability to undergoimmunoglobulin class switch and is associated with hyper-IgM syndrome.

Contacting: Placement in direct physical association; includes both insolid and liquid form.

Diagnosis: The process of identifying a disease by its signs, symptomsand results of various tests. The conclusion reached through thatprocess is also called “a diagnosis.” Forms of diagnostic testingcommonly performed include blood tests, medical imaging, geneticanalysis, molecular marker analysis, urinalysis, biopsy and histology.Diagnostic methods differ in their sensitivity and specificity. The“sensitivity” of a diagnostic assay is the percentage of diseasedindividuals (for example, individuals undergoing organ transplantrejection) who test positive (percent of true positives). The“specificity” of a diagnostic assay is 1 minus the false positive rate,where the false positive rate is defined as the proportion of thosewithout the disease who test positive. While a particular diagnosticmethod may not provide a definitive diagnosis of a condition, it iseffective if the method provides a positive indication that aids indiagnosis. “Prognostic” is the probability of development (or forexample, the probability of severity) of a pathologic condition, such asorgan rejection.

Fluorophore: A chemical compound, which when excited by exposure to aparticular stimulus, such as a defined wavelength of light, emits light(fluoresces), for example at a different wavelength (such as a longerwavelength of light).

Fluorophores are part of the larger class of luminescent compounds.Luminescent compounds include chemiluminescent molecules, which do notrequire a particular wavelength of light to luminesce, but rather use achemical source of energy. Therefore, the use of chemiluminescentmolecules (such as aequorin) can eliminate the need for an externalsource of electromagnetic radiation, such as a laser.

Examples of particular fluorophores that can be used in the disclosedmethods are provided in U.S. Pat. No. 5,866,366 to Nazarenko et al.(which is hereby incorporated by reference in its entirety), such as4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine andderivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BrilliantYellow, coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine;IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone;ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (Cibacron™ Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acidand terbium chelate derivatives; LightCycler Red 640; Cy5.5; andCy56-carboxyfluorescein; 5-carboxyfluorescein (5-FAM); borondipyrromethene difluoride (BODIPY);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); acridine, stilbene,-6-carboxy-fluorescein (HEX), TET (Tetramethyl fluorescein),6-carboxy-X-rhodamine (ROX), Texas Red,2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE), Cy3, Cy5,VIC® (Applied Biosystems), LC Red 640, LC Red 705, Yakima yellow amongstothers.

Other suitable fluorophores include those known to those skilled in theart, for example those available from Molecular Probes (Eugene, Oreg.).In particular examples, a fluorophore iscarbosyflouresciensuccinimyldiester.

Graft-Versus-Host Disease (GVHD): A common and serious complication ofbone marrow or other tissue transplantation wherein there is a reactionof donated immunologically competent lymphocytes against a transplantrecipient's own tissue. GVHD is a possible complication of anytransplant that uses or contains stem cells from either a related or anunrelated donor.

There are two kinds of GVHD, acute and chronic. Acute GVHD appearswithin the first three months following transplantation. Signs of acuteGVHD include a reddish skin rash on the hands and feet that may spreadand become more severe, with peeling or blistering skin. Acute GVHD canalso affect the stomach and intestines, in which case cramping, nausea,and diarrhea are present. Yellowing of the skin and eyes (jaundice)indicates that acute GVHD has affected the liver. Chronic GVHD is rankedbased on its severity: stage/grade 1 is mild; stage/grade 4 is severe.Chronic GVHD develops three months or later following transplantation.The symptoms of chronic GVHD are similar to those of acute GVHD, but inaddition, chronic GVHD may also affect the mucous glands in the eyes,salivary glands in the mouth, and glands that lubricate the stomachlining and intestines.

IgA: An antibody isotype that plays a critical role in mucosal immunity.More IgA is produced in mucosal linings than all other types of antibodycombined. There are two subclasses of IgA (IgA1 and IgA2) and can existin a dimeric form called secretory IgA (sIgA). In its secretory form,IgA is the main immunoglobulin found in mucous secretions, includingtears, saliva, colostrum and secretions from the genito-urinary tract,gastrointestinal tract, prostate and respiratory epithelium. It is alsofound in small amounts in blood. The secretory component of sIgAprotects the immunoglobulin from being degraded by proteolytic enzymes,thus sIgA can survive in the harsh gastrointestinal tract environmentand provide protection against microbes that multiply in bodysecretions.

IgD: An antibody isotype that makes up about 1% of proteins in theplasma membranes of immature B-lymphocytes where it is usuallycoexpressed with another cell surface antibody called IgM. IgD is alsoproduced in a secreted form that is found in very small amounts in bloodserum. Secreted IgD is produced as a monomeric antibody with two heavychains of the delta (δ) class, and two immunoglobulin light chains.

IgM: An antibody isotype that is present on B cells. IgM is the largesttype of antibody molecule in the human circulatory system. It isproduced after an animal has been exposed to an antigen for an extendedtime or when an animal is exposed to an antigen for the second time.

Immune response: A response of a cell of the immune system, such as a Bcell, or a T cell, to a stimulus. In one embodiment, the response isspecific for a particular antigen (an “antigen-specific response”).

A “parameter of an immune response” is any particular measurable aspectof an immune response, including, but not limited to, cytokine secretion(IL-6, IL-10, IFN-γ, etc.), immunoglobulin production, dendritic cellmaturation, and proliferation of a cell of the immune system. One ofskill in the art can readily determine an increase in any one of theseparameters, using known laboratory assays. In one specific non-limitingexample, to assess cell proliferation, incorporation of ³H-thymidine canbe assessed. A “substantial” increase in a parameter of the immuneresponse is a significant increase in this parameter as compared to acontrol. Specific, non-limiting examples of a substantial increase areat least about a 50% increase, at least about a 75% increase, at leastabout a 90% increase, at least about a 100% increase, at least about a200% increase, at least about a 300% increase, and at least about a 500%increase. Similarly, an inhibition or decrease in a parameter of theimmune response is a significant decrease in this parameter as comparedto a control. Specific, non-limiting examples of a substantial decreaseare at least about a 50% decrease, at least about a 75% decrease, atleast about a 90% decrease, at least about a 100% decrease, at leastabout a 200% decrease, at least about a 300% decrease, and at leastabout a 500% decrease. A statistical test, such as a non-paramentricANOVA, can be used to compare differences in the magnitude of theresponse induced by one agent as compared to the percent of samples thatrespond using a second agent. In some examples, p≤0.05 is significant,and indicates a substantial increase or decrease in the parameter of theimmune response. One of skill in the art can readily identify otherstatistical assays of use.

Immunocompromised subject: A subject who is incapable of developing orunlikely to develop a robust immune response, usually as a result ofdisease, malnutrition, or immunosuppressive therapy. Animmunocompromised immune system is an immune system that is functioningbelow normal. Immunocompromised subjects are more susceptible toopportunistic infections, for example viral, fungal, protozoan, orbacterial infections, prion diseases, and certain neoplasms. Those whocan be considered to be immunocompromised include, but are not limitedto, subjects with AIDS (or HIV positive), subjects with severe combinedimmune deficiency (SCID), diabetics, subjects who have had transplantsand who are taking immunosuppressives, and those who are receivingchemotherapy for cancer. Immunocompromised individuals also includessubjects with most forms of cancer (other than skin cancer), sickle cellanemia, cystic fibrosis, those who do not have a spleen, subjects withend stage kidney disease (dialysis), and those who have been takingcorticosteroids on a frequent basis by pill or injection within the lastyear. Subjects with severe liver, lung, or heart disease also may beimmunocompromised.

Infectious disease: Any disease caused by an infectious agent. Examplesof infectious pathogens include, but are not limited to: viruses,bacteria, mycoplasma and fungi. In a particular example, it is a diseasecaused by at least one type of infectious pathogen. In another example,it is a disease caused by at least two different types of infectiouspathogens. Infectious diseases can affect any body system, be acute(short-acting) or chronic/persistent (long-acting), occur with orwithout fever, strike any age group, and overlap each other. Infectiousdiseases can be opportunistic infections, in that they occur morefrequently in immunocompromised subjects.

Viral diseases commonly occur after immunosuppression due tore-activation of viruses already present in the recipient. Particularexamples of viral infections include, but are not limited to,cytomegalovirus (CMV) pneumonia, enteritis and retinitis; Epstein-Barrvirus (EBV) lymphoproliferative disease; chicken pox/shingles (caused byvaricella zoster virus, VZV); HSV-1 and -2 mucositis; HSV-6encephalitis, BK-virus hemorrhagic cystitis; viral influenza; pneumoniafrom respiratory syncytial virus (RSV); AIDS (caused by HIV); andhepatitis A, B or C. Opportunistic infections occur in a subject with acompromised immune system, such as a subject who has beenimmuno-depleted and recently received a bone marrow transplant or ahematopoietic stem cell transplant. These infections include, but arenot limited to cytomegalovirus, Candida albicans, human immunodeficiencyvirus, Staphlococcus aureus, Steptococcus pyogenes, Pseudomasaeruginosa, Acinteobacter baumanni, Toxoplasma gondii, Pneumocystitiscarinii, or Aspergillus infections.

Additional examples of infectious virus include: Retroviridae;Picornaviridae (for example, polio viruses, hepatitis A virus;enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (such as strains that cause gastroenteritis); Togaviridae(for example, equine encephalitis viruses, rubella viruses); Flaviridae(for example, dengue viruses, encephalitis viruses, yellow feverviruses); Coronaviridae (for example, coronaviruses); Rhabdoviridae (forexample, vesicular stomatitis viruses, rabies viruses); Filoviridae (forexample, ebola viruses); Paramyxoviridae (for example, parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (for example, influenza viruses); Bungaviridae (forexample, Hantaan viruses, bunga viruses, phleboviruses and Nairoviruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g.,reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae(Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae(papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses);Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella zostervirus, cytomegalovirus (CMV), herpes viruses); Poxviridae (variolaviruses, vaccinia viruses, pox viruses); and Iridoviridae (such asAfrican swine fever virus); and unclassified viruses (for example, theetiological agents of Spongiform encephalopathies, the agent of deltahepatitis (thought to be a defective satellite of hepatitis B virus),the agents of non-A, non-B hepatitis (class 1=internally transmitted;class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk andrelated viruses, and astroviruses).

Examples of fungal infections include but are not limited to:aspergillosis; thrush (caused by Candida albicans); cryptococcosis(caused by Cryptococcus); and histoplasmosis. Thus, examples ofinfectious fungi include, but are not limited to, Cryptococcusneoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomycesdermatitidis, Chlamydia trachomatis, Candida albicans.

Examples of infectious bacteria include: Helicobacter pyloris, Boreliaburgdorferi, Legionella pneumophilia, Mycobacteria sps (such as. M.tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillusanthracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira, andActinomyces israelli. Other infectious organisms (such as protists)include: Plasmodium falciparum and Toxoplasma gondii.

Isolated: An “isolated” biological component (such as a nucleic acid,peptide or protein) has been substantially separated, produced apartfrom, or purified away from other biological components in the cell ofthe organism in which the component naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins which have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids. Similarly, an “isolated” cell has been substantiallyseparated, produced apart from, or purified away from other cells of theorganism in which the cell naturally occurs. Isolated cells can be, forexample, at least 99%, at least 98%, at least 95%, at least 90%, atleast 85%, or at least 80% pure.

Label: An agent capable of detection, for example by ELISA,spectrophotometry, mass spectrometry, flow cytometry, or microscopy. Forexample, a label can be attached to a molecule, thereby permittingdetection of the molecule. In one particular example, a label isattached to an antibody. Examples of labels include, but are not limitedto, radioactive isotopes, enzyme substrates, co-factors, ligands,chemiluminescent agents, fluorophores, haptens, enzymes, metals, metalisotopes and combinations thereof. Methods for labeling and guidance inthe choice of labels appropriate for various purposes are discussed forexample in Sambrook et al. (Molecular Cloning: A Laboratory Manual, ColdSpring Harbor, New York, 1989) and Ausubel et al. (In Current Protocolsin Molecular Biology, John Wiley & Sons, New York, 1998). In aparticular example, donor and third-party antigens are labeled with afluorophore, such as carboxyflouresciensuccinimyldiester to allowantigen detection by flow cytometry.

Leukocyte: Cells in the blood, also termed “white cells,” that areinvolved in defending the body against infective organisms and foreignsubstances. Leukocytes are produced in the bone marrow. There are 5 maintypes of white blood cell, subdivided between 2 main groups:polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) andmononuclear leukocytes (monocytes and lymphocytes).

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. The main types of lymphocytes are: B cells, Tcells and natural killer cells (NK cells). “T lymphocytes” or “T cells”are non-antibody producing lymphocytes that constitute a part of thecell-mediated arm of the immune system. T cells arise from immaturelymphocytes that migrate from the bone marrow to the thymus, where theyundergo a maturation process under the direction of thymic hormones.Here, the mature lymphocytes rapidly divide increasing to very largenumbers. The maturing T cells become immunocompetent based on theirability to recognize and bind a specific antigen. Activation ofimmunocompetent T cells is triggered when an antigen binds to thelymphocyte's surface receptors. T cells include, but are not limited to,CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ T lymphocyte is an immune cellthat carries a marker on its surface known as “cluster ofdifferentiation 4” (CD4). These cells, also known as helper T cells,help orchestrate the immune response, including antibody responses aswell as killer T cell responses. CD8⁺ T cells carry the “cluster ofdifferentiation 8” (CD8) marker. In one embodiment, a CD8 T cell is acytotoxic T lymphocyte. In another embodiment, a CD8 cell is asuppressor T cell.

Major Histocompatability Complex (MHC): A generic designation meant toencompass the histocompatability antigen systems described in differentspecies, including the human leukocyte antigens (“HLA”).

Monocyte: A type of mononuclear leukocyte. Monocytes are produced by thebone marrow from hematopoietic stem cell precursors called monoblasts.Monocytes circulate in the bloodstream for about one to three days andthen typically move into tissues throughout the body. Monocytes whichmigrate from the bloodstream to other tissues will then differentiateinto tissue resident macrophages or dendritic cells. They constitutebetween three to eight percent of the leukocytes in the blood. Monocytesand monocyte-derived cells such as dendritic cells have three mainfunctions in the immune system: phagocytosis, antigen presentation andcytokine production. Thus, monocytes and monocyte-derived dendriticcells are APCs. Monocyte cells express CD14. Some types of monocytesexpress CD16 in addition to CD14.

Organ rejection or transplant rejection: Functional and structuraldeterioration of an organ due to an active immune response expressed bythe recipient, and independent of non-immunologic causes of organdysfunction.

Sample (or biological sample): A biological specimen containing genomicDNA, RNA (including mRNA and microRNA), protein, cells, tissues orcombinations thereof, obtained from a subject. Examples include, but arenot limited to, peripheral blood (including monocytes ormonocyte-derived cells, such as dendritic cells), urine, saliva, tissuebiopsy, fine needle aspiration samples, surgical specimen, and autopsymaterial. In one example, a sample is blood sample which includeslymphocytes, leukocytes, such as peripheral blood leukocytes, or acombination thereof with or without red blood cells.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals. In one particular example, thesubject is a child. As used herein, a “child” refers to a person underthe age of 18.

T-Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, also known ashelper T cells, help orchestrate the immune response, including antibodyresponses as well as killer T cell responses. CD8⁺ T cells carry the“cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8 Tcells is a cytotoxic T lymphocytes. In another embodiment, a CD8 cell isa suppressor T cell.

Therapeutic agent: A chemical compound, small molecule, or othercomposition, such as an antisense compound, antibody, proteaseinhibitor, hormone, chemokine, cytokine, radioactive agent, oranti-inflammatory agent, capable of inducing a desired therapeutic orprophylactic effect when properly administered to a subject.

Therapeutically effective amount: A quantity of a specifiedpharmaceutical or therapeutic agent sufficient to achieve a desiredeffect in a subject, or in a cell, being treated with the agent. Theeffective amount of the agent will be dependent on several factors,including, but not limited to the subject or cells being treated, andthe manner of administration of the therapeutic composition.

Under conditions sufficient to: A phrase that is used to describe anyenvironment that permits the desired activity. In one example, includesconditions sufficient to induce uptake of a molecule, such as thebinding or internalization of an antigen by a cell (e.g., the bindingand/or internalization of donor antigen by an APC.

III. Methods for Diagnosing or Predicting Organ Transplant Rejection orGVHD

Disclosed herein is an assay for diagnosis and prediction of transplantrejection, such as the acute cellular rejection (ACR) or humoralrejection (HR) of an organ transplant or graft-versus-host disease. Insome examples, this assay exploits the fact that a B-cell is a potentAPC. Monitoring antigen uptake and presentation by the APCs (such asB-cells, monocytes or monocyte-derived cells) serves as an early warningfor whether the effector response favors ACR, or HR, or a rejection-freestate. The disclosed antigen presenting test has several uses intransplantation. In some examples, the assay is used to measure the riskof rejection to manage anti-rejection drug therapy. In some examples,the assay is used to calculate the time to reduction of rejection-riskor API<1 when estimating time to reduction of immunosuppressive drugsincluding but not limited to steroids, tacrolimus, etc. or comparing twodifferent anti-rejection drug regimens, or two different types oftransplant recipients. In some examples, the assay is used to estimatethe severity of rejection. For example, the API is higher with rejectionof higher severity which requires more intensive anti-rejection drugtherapy. In some examples, the disclosed assay is used to estimating thelikelihood of graft loss. In some examples, the disclosed assay is usedto establish a personalized rejection risk estimate at any given timefrom an individual recipient in which the same donor or surrogate donorstimulator is used, and the same third-party antigen is used as areference for donor antigen presentation for the same transplantrecipient. In some examples, the disclosed assay is used in combinationa variety of markers, which are unique to the different developmentalstages of the B-cell. B-cell antigen presentation can be used todetermine when naïve B-cells have transitioned into memory IgG+B-cell.This transition from naïve to memory IgG+B-cells is characterized by thedecrease or loss of antigen presenting function. For this utility,several B-cell lineage-specific markers can be used. In some examples,the disclosed assay is used in combination with at least one marker of amonocyte, such as CD14.

In some embodiments, the subject is a human. In particular examples, thesubject is a human child, such as a child of zero to five years of age,less 10 years of age, less than 13 years of age, or less than 18 yearsof age. In other embodiments, the subject is an adult subject, such as asubject greater than 18 years of age, greater than 20 years of age orgreater than 25 years of age. In other embodiments, the subject is anon-human animal, such as a veterinary subject (for example, a small orlarge domestic animal).

The presently disclosed assay has significantly higher sensitivity andspecificity than previously disclosed assays. For example, previousstudies have evaluated B-cell and other APC functions by incubating APCswith a non-specific antigen, ovalbumin (OVA) in which OVA uptake wasused as a measure of antigen presentation. The sensitivity andspecificity of B-cell OVA uptake was 70-75% for association withrejection-prone (Rejector) status. However, for clinical implementation,a more sensitive and specific test is needed.

In some examples, a method of assessing organ rejection is disclosed. Itis contemplated that the transplant can be any organ, including solidorgans. In some examples, the subject has received the transplant. Insome examples, the subject is a candidate to receive the transplant.Examples of solid organs include, but are not limited to, liver,intestine, kidney, heart, lung, pancreas and skin. In the context of thepresent disclosure, a transplanted organ need not be the entire organ,but can be a portion or section of the organ. In particular examples,the subject has received, or is in need or receiving, multiple organs,or portions of multiple organs. In some cases, the subject is atransplant recipient or candidate for a transplant of a combination oftwo or more of a solid organ, bone marrow and stem cells. In some cases,the subject is undergoing treatment, including immunosuppressivetherapy.

In some examples, the method includes contacting a first samplecomprising APCs obtained from a subject in need of or having received anorgan transplant from a donor, with a donor antigen from the donor,under conditions sufficient to induce uptake of the donor antigen;contacting a second sample comprising APCs obtained from the subject inneed of or having received an organ transplant, with a third-partyantigen, under conditions sufficient to induce uptake of the third-partyantigen; and determining the ratio of uptake of the donor antigen in thefirst sample to uptake of the third-party antigen in the second sample.A ratio of greater than one indicates organ rejection in the subject ora predisposition for organ rejection. In some embodiments, the APCs areB cells. In some embodiments, the APCs are monocytes or monocyte derivedcells, such as monocyte-derived dendritic cells.

In some embodiments, methods are for assessing rejection of a solidorgan transplant. These assays measure the uptake of donor antigen andexpress it as a ratio with uptake of third-party antigen in APCs, suchas B cells, monocytes or monocyte derived cells, such asmonocyte-derived dendritic cells. This ratio is the API. An API>1indicates increased risk of rejection or the presence of rejection. Forexample, and API of greater than 1.2, greater than 1.5, greater than1.75, greater than 2, greater than 3, greater than 4, greater than 5,greater than 6, greater than 7, greater than 8, greater than 9, orgreater than 10, such as between 1.2-10, 5-10, 1.2-3, 1.5-2.5, including1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4. 4.5, 5, 5.5,6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 or more can indicate increased riskof rejection. An API<1 indicates decreased risk of rejection orrejection. For example, and API of less than 0.9, less than 0.8, lessthan 0.75, less than 0.6, less than 0.5, less than 0.1 or less than0.01, such as between 0.2 and 0.9, 0.3 and 0.8, 0.4 and 0.7, 0.5 and0.6, including 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.60, 0.65, 0.7,0.75, 0.8, 0.85, 0.9, 0.95 can indicate a decreased risk of rejection.The API is unique in that it “normalizes” donor antigen uptake to theuptake of a reference (third-party) antigen for each person. In thisway, the API is unique for each individual, and yet comparable betweenindividuals, on the basis of this normalized scale.

Also provided herein is a method of assessing GVHD. In some examples,the method includes contacting a first sample comprising APCs obtainedfrom a sample of donor bone marrow or stem cells before transplantation,or from the recipient who has received donor bone marrow or stem cells.The method includes contacting APCs from the subject followingtransplantation, with a recipient antigen from the recipient, underconditions sufficient to induce uptake of the recipient antigen;contacting a second sample comprising APCs obtained from a sample ofdonor bone marrow or stem cells before transplantation, or from therecipient who has received donor bone marrow or stem cells, with athird-party antigen, under conditions sufficient to induce uptake of thethird-party antigen; and determining the ratio of uptake of therecipient antigen in the first sample to uptake of the third-partyantigen in the second sample. A ratio of greater than one indicates GVHDin the subject or a predisposition for GVHD. In some embodiments, theAPCs are B cells, monocytes or monocyte derived cells, such asmonocyte-derived dendritic cells.

In some embodiments, the methods for assessing GVHD include measuringthe uptake of recipient antigen and expressing it as a ratio with uptakeof third-party antigen in APCs, such as B cells, monocytes or monocytederived cells, such as monocyte-derived dendritic cells, to determinethe API. An API>1 indicates increased risk of GVHD or the presence ofGVHD. For example, and API of greater than 1.2, greater than 1.5,greater than 1.75, greater than 2, greater than 3, greater than 4,greater than 5, greater than 6, greater than 7, greater than 8, greaterthan 9, or greater than 10, such as between 1.2-10, 5-10, 1.2-3,1.5-2.5, including 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3,3.5, 4. 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 or more canindicate increased risk of GVHD. An API<1 indicates decreased risk ofGVHD or GVHD. For example, an API of less than 0.9, less than 0.75, lessthan 0.5, less than 0.1 or less than 0.01, such as between 0.2 and 0.9,0.3 and 0.8, 0.4 and 0.7, 0.5 and 0.6, including 0.01, 0.02, 0.03, 0.04,0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4,0.45, 0.5, 0.55, 0.60, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 canindicate decreased risk of GVHD.

In some examples, the disclosed assays predict B-cell rejection with asensitivity of at least 90% and a specificity of at least 90% for anincreased risk of B-cell rejection or the presence of B-cell rejection.In some examples, the methods disclosed herein have a sensitivity of atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%,including between 90% to 98%, between 92% to 96%, between 92% to 95%,between 93% and 95%, between 94% and 96%, including 90%, 91%, 92,%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sensitivity. In some examples, themethods disclosed herein have a specificity of at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98% or at least 99%, including between 90%to 98%, between 92% to 96%, between 92% to 95%, between 93% and 95%,between 94% and 96%, including 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%,98%, or 99% specificity.

In some examples, the disclosed assays predict ACR and/or HR rejectionwith a sensitivity of at least 75% and a specificity of at least 90% foran increased risk of ACR and/or HR rejection or the presence of ACRand/or HR rejection. In some examples, the methods disclosed herein havea sensitivity of at least 75%, at least 76%, at least 77%, at least 78%,at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99%, including between 75% to 98%, between 75% to 80%, between 90%to 98%, between 92% to 96%, between 92% to 95%, between 93% and 95%,between 94% and 96%, including 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92,%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sensitivity. In some examples, the methods disclosedherein have a specificity of at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99%, including between 90% to 98%, between 92% to96%, between 92% to 95%, between 93% and 95%, between 94% and 96%,including 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%specificity.

In some embodiments of the method, an API of greater than one predictsrejection with a sensitivity of approximately 94%, 95%, 96%, 97%, 98% or99% and/or a specificity of approximately 94%, 95%, 96%, 97%, 98% or99%.

In some embodiments, comparing uptake of the donor (or recipientantigen) to uptake of the third party antigen further includes labelinga biological sample comprising donor (or recipient) antigen and abiological sample comprising third party antigen with a detectablelabel. Any type of detectable label can be used to facilitate detection.Specific, non-limiting examples of labels include fluorescent tags,enzymes, and radioactive isotopes. Fluorophores are part of the largerclass of luminescent compounds. Luminescent compounds includechemiluminescent molecules, which do not require a particular wavelengthof light to luminesce, but rather use a chemical source of energy.Therefore, the use of chemiluminescent molecules (such as aequorin) caneliminate the need for an external source of electromagnetic radiation,such as a laser.

Examples of particular fluorophores that can be used in the methodsdisclosed herein are provided in U.S. Pat. No. 5,866,366 to Nazarenko etal., such as 4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid,acridine and derivatives such as acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BrilliantYellow, coumarin and derivatives such as coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;4′,6-diaminidino-2-phenylindole (DAPI);5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives such as eosin and eosin isothiocyanate; erythrosin andderivatives such as erythrosin B and erythrosin isothiocyanate;ethidium; fluorescein and derivatives such as 5-carboxyfluorescein(FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), and QFITC (XRITC); fluorescamine;IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone;ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such aspyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red4 (Cibacron™ Brilliant Red 3B-A); rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acidand terbium chelate derivatives; LightCycler Red 640; Cy5.5; andCy56-carboxyfluorescein; 5-carboxyfluorescein (5-FAM); borondipyrromethene difluoride (BODIPY);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); acridine, stilbene,-6-carboxy-fluorescein (HEX), TET (Tetramethyl fluorescein),6-carboxy-X-rhodamine (ROX), Texas Red,2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE), Cy3, Cy5,VIC® (Applied Biosystems), LC Red 640, LC Red 705, Yakima yellow amongstothers.

Other suitable fluorophores include those known to those skilled in theart, for example those available from Molecular Probes (Eugene, Oreg.).In some examples, the fluorescent molecule iscarboxyfluoresceinsuccinimidyl ester or other like compounds.

The method includes determining the ratio of uptake of the donor (orrecipient) antigen in the first sample to uptake of the third-partyantigen in the second sample by measuring the fluorescence of the twosamples.

In one embodiment, uptake can be measured by fluorescent activated cellsorting (FACS). FACS employs a plurality of color channels, low angleand obtuse light-scattering detection channels, and impedance channels,among other more sophisticated levels of detection, to separate or sortcells. Any FACS technique may be employed as long as it is notdetrimental to the viability of the desired cells (for exemplary methodsof FACS see U.S. Pat. No. 5,061,620, herein incorporated by reference).

For example, the sample including APCs is obtained from blood, spleen orbone marrow. In some embodiments, the first sample and/or the secondsample that contain APCs are peripheral blood lymphocytes or peripheralblood leukocytes (including monocytes or monocyte-derived cells, such asdendritic cells).

The antigen can be a purified or isolated antigen. Thus, the antigen canbe synthesized or produced by molecular biology techniques. The donorantigen can be any antigen of interest from the donor. One isolatedantigen, or more than one isolated antigen, such as at least two, atleast three, at least four, at least 5, at least 10 or more isolatedantigens can be utilized.

In some examples, the antigen is included in a complex biologicalsample, such as a cell lysate or fraction. In some examples, the antigenis a cell lysate of lymphocytes and/or leukocytes. In some embodiments,the donor antigen or the third-party antigen include donor cells, anantigenic peptide, an antigenic peptide labeled with a fluorochrome, orany combination thereof.

In some embodiments, for assessing transplant rejection, one antigen atthe HLA-A, HLA-B or HLA-DR loci from the donor is utilized in themethod. In some embodiments, more than one antigen at the HLA-A, HLA-Bor HLA-DR loci from the donor is utilized in the method. In furtherembodiments, a combination of antigens from the HLA-A, HLA-B and HLA-DRloci are utilized in the methods. In one example, the donor antigenincludes antigens from lymphocytes, leukocytes, such as peripheral bloodleukocytes (including monocytes or monocyte-derived cells, such asdendritic cells) or a combination thereof. In some examples, donorantigen includes lysed cell membranes from donor peripheral bloodleukocytes, spleen cells, or bone marrow cells. In an example, donorantigen is provided from a subject that has the same or very similarHLA-A, HLA-B, or HLA-DR loci profile as the donor, but is not the donor.

In other embodiments, for assessing GVHD, one antigen at the HLA-A,HLA-B or HLA-DR and other HLA loci from the recipient is utilized in themethod. In other embodiments, more than one antigen at the HLA-A, HLA-Bor HLA-DR loci from the recipient is utilized in the method to detectGVHD. In further embodiments, a combination of antigens from the HLA-A,HLA-B and HLA-DR and other HLA loci are utilized in this method. In oneexample, the recipient antigen includes antigens from lymphocytes,leukocytes, such as peripheral blood leukocytes (including monocytes ormonocyte-derived cells, such as dendritic cells) or a combinationthereof. In some examples, recipient antigen includes lysed cellmembranes from recipient peripheral blood leukocytes, spleen cells, orbone marrow cells. In an example, recipient antigen is provided from asubject that had the same of very similar HLA-A, HLA-B, or HLA-DR andother HLA loci profile as the recipient, but is not the recipient. Inone example, minor HLA antigens other than or in addition to HLA-A, -Band -DR are utilized.

The third party antigen can be from any subject who is allogeneic toboth the donor and the recipient. One antigen or more than one antigencan be utilized. In some embodiments, one antigen at the HLA-A, HLA-B orHLA-DR and other HLA loci from a third party is utilized in the method.In other embodiments, more than one antigen at the HLA-A, HLA-B orHLA-DR and other HLA loci from the third party is utilized in themethod. In further embodiments, a combination of antigens from theHLA-A, HLA-B and HLA-DR and other HLA loci are utilized in the methods.Exemplary third-party antigen samples also include lymphocytes,leukocytes, such as peripheral blood leukocytes (including monocytes ormonocyte-derived cells, such as dendritic cells) or a combinationthereof. For example, third-party antigen samples include lysed cellmembranes from third party peripheral blood leukocytes, spleen cells, orbone marrow cells.

In some embodiments, the method is used to titrate the dose of animmunosuppressive agent provided to the subject or evidence theeffectiveness of an immunosuppressive regimen for the treatment oftransplant rejection. For example, a subject is given a first treatmentwith an immunosuppressive regimen. A first sample including APCs, suchas B cells, monocytes or monocyte-derived cells (such as dendriticcells), is obtained from a subject having received an organ transplantfrom a donor is contacted with a donor antigen from the donor, underconditions sufficient to induce uptake of the donor antigen. A secondsample including APCs, such as B cells monocytes or monocyte-derivedcells (such as dendritic cells), obtained from the subject is contactedwith a third-party antigen, under conditions sufficient to induce uptakeof the third-party antigen. The ratio of uptake of the donor antigen inthe first sample to uptake of the third-party antigen in the secondsample is determined. A ratio of greater than one indicates organrejection in the subject and indicates that immunosuppression should beincreased, or that a different immunosuppressive regimen should be used.A ratio of less than one indicates the absence of organ rejection in thesubject and indicates that immunosuppression can be maintained ordecreased, or indicates that the immunosuppressive regimen isappropriate for the subject. The methods can be repeated, so that thesubject is monitored regularly. For example, the method can be repeateddaily, bi-weekly, weekly, bi-monthly, or monthly. The immunosuppressiveagent can include, but is not limited to, a steroid, cyclosporine A,anti-CD3, anti-CD25 (such as daclizumbab), cytokines, rapamycin, or anyother immunosuppressive agent of interest including but not limited totacrolimus, bortezimib, alemtuzumab, anti-human thymocyte globulin,anti-lymphocyte globulin, mycophenolate mofetil, etc.

In some embodiments, the method is used to assess the degree of organrejection. For example, a higher API is associated with more severerejection than with mild rejection. To illustrate further, a higher APIis associated with steroid-resistant than with steroid-sensitiverejection.

In some embodiments, the method is used to titrate the dose of animmunosuppressive agent provided to the subject or evidence theeffectiveness of an immunosuppressive regimen for treatment of GVHD. Forexample, a subject is given a first treatment with an immunosuppressiveregimen. A first sample comprising APCs, such as B cells, monocytes ormonocyte-derived cells (such as dendritic cells), is obtained from asubject having received an organ transplant from a donor is contactedwith a recipient antigen from the recipient, under conditions sufficientto induce uptake of the recipient antigen. A second sample comprisingAPCs, such as B cells, monocytes or monocyte-derived cells (such asdendritic cells), obtained from the subject is contacted with athird-party antigen, under conditions sufficient to induce uptake of thethird-party antigen. The ratio of uptake of the recipient antigen in thefirst sample to uptake of the third-party antigen in the second sampleis determined. A ratio of greater than one indicates GVHD in the subjectand indicates that immunosuppression should be increased, or that adifferent immunosuppressive regimen should be used. A ratio of less thanone indicates the absence of GVHD in the subject and indicates thatimmunosuppression can be maintained or decreased, or indicates that theimmunosuppressive regimen is appropriate for the subject. API numbers<0.9, or <0.1 indicate decreased risk, while API>1.2 or >2 or >3 wouldindicate increased risk of GVHD. The methods can be repeated, so thatthe subject is monitored regularly. For example, the method can berepeated daily, bi-weekly, weekly, bi-monthly, or monthly. Theimmunosuppressive agent can include, but is not limited to, a steroid,cyclosporine A, anti-CD4, anti-CD25 (such as daclizumbab), cytokines,rapamycin, or any other immunosuppressive agent of interest includingbut not limited to tacrolimus, bortezimib, alemtuzumab, anti-humanthymocyte globulin, anti-lymphocyte globulin, mycophenolate mofetil,etc.

In some embodiments of the method, for detecting transplant rejection,determining the ratio of uptake of the donor antigen in the first sampleto uptake of the third-party antigen in the second sample includesdetecting a plurality of biomarkers following treatment with the donorantigen with the first sample comprising antigen presenting cells andthe third-party antigen with the second sample comprising antigenpresenting cells, and comparing the expression of the plurality ofbiomarkers following treatment with donor antigen to the expression ofthe biomarkers following treatment with the third-party antigen todetermine the API. For example, markers categorize a B-cell as memory ornaive or as plasma cell precursors, based on whether they express CD27,IgA, IgM, IgG, IgD, CD25, CD5, CD10, CD154, CD138, CD19, CD38, CD24,CTLA4, etc. Further, CD14 and/or CD16 markers identify monocytes. Inother embodiments, for detecting GVHD, determining the ratio of uptakeof the recipient antigen in the first sample to uptake of thethird-party antigen in the second sample includes detecting a pluralityof biomarkers on the antigen presenting cells following treatment withthe recipient antigen with the first sample comprising antigenpresenting cells and the third-party antigen with the second samplecomprising antigen presenting cells, and comparing the expression of theplurality of biomarkers following treatment with recipient antigen tothe expression of the biomarkers following treatment with thethird-party antigen to determine the antigen presenting index.

In some examples, the plurality of biomarkers includes at least one ofCD27, IgM, IgA, IgD, CD5, CD10, such as one, two, three, four, five orall six markers. In other examples, additional biomarkers such memorycell markers, plasma cell markers, B-cell activation markers,leukocyte/lymphocyte markers (such as CD14 and/or CD16 for monocytemarkers), cell viability markers, and other markers known to those ofskill in the art to be useful in monitoring organ transplant rejection.In some examples, additional markers included IgG (memory cell marker),CD19, CD38 (plasma cell marker), CD138 (plasma cell activation markers),CD154 (B-cell activation marker), CTLA4 (negative B-cell co-stimulatormarker), CD45 (pan-leukocyte/lymphocyte marker), CD14 and/or CD16(monocyte markers) or any combination thereof. Thus, the method can alsoinclude measuring B cells, T cells and/or monocytes, such as memory Tcells that express CTLA4 or CD154, B cells that express CD154+CD19+and/or monocytes that express CD14 and/or CD16 cells.

In some embodiments of the method, for detecting transplant rejection,determining the ratio of uptake of the donor antigen in the first sampleto uptake of the third-party antigen in the second sample includesdetecting at least one biomarker following treatment with the donorantigen with the first sample comprising antigen presenting cells andthe third-party antigen with the second sample comprising antigenpresenting cells, and comparing the expression of the at least onebiomarker following treatment with donor antigen to the expression ofthe biomarkers following treatment with the third-party antigen todetermine the API, wherein the at least one biomarker is CD14 and theAPCs are monocytes or monocyte-derived cells. It is contemplated thatadditional biomarkers include at least one of CD16, CD27, IgM, IgA, IgD,CD5, CD10, such as one, two, three, four, five or all six markers inaddition to CD14. In other examples, additional biomarkers to monocytebiomarkers (CD14 and/or CD16) include markers such as memory cellmarkers, plasma cell markers, B-cell activation markers,leukocyte/lymphocyte markers, cell viability markers, and other markersknown to those of skill in the art to be useful in monitoring organtransplant rejection. In some examples, additional markers include IgG(memory cell marker), CD19, CD38 (plasma cell marker), CD138 (plasmacell activation markers), CD154 (B-cell activation marker), CTLA4(negative B-cell co-stimulator marker), CD45 (pan-leukocyte/lymphocytemarker), or any combination thereof. Thus, the method can also includemeasuring B cells, T cells and/or monocytes, such as memory T cells thatexpress CTLA4 or CD154, B cells that express CD154+CD19+ and/ormonocytes that express CD14 and/or CD16 cells.

In one embodiment, the method includes assessing the API and the numberof inflammatory donor-specific B cells, which express CD154. In otherexample, the method includes assessing the API and the number of CTLA4+T-cytotoxic memory T cells. In a further example, the method includesmeasuring the API and the number of CD154+CD19+ B cells. In one example,API is positively correlated with inflammatory donor specific B cells,which express CD154 and CD19. As such, an API greater than one indicatesincreased CD154+ and CD19 cells in the recipient sample. In anotherexample, API is negatively correlated with anti-inflammatory donorspecific T cytotoxic memory cells expressing CTLA4. In this example, anAPI greater than one indicates fewer CTLA4 T cells in the donor sample.In a further example, API is negatively correlated withanti-inflammatory donor specific B cells expressing the marker CTLA4 inwhich an API index greater than one indicates fewer CTLA4 B cells in thedonor sample. The method can include measuring one or more of these celltypes. In other example, the method includes assessing the API and thenumber of CD14+ cells.

In some examples, the plurality of biomarkers includes at least CD27,IgM, IgA, IgD, IgG, CD5 and CD10. It is contemplated that additionalbiomarkers can also be detected, such as early B-cell lineage markers,CD24, and CD179b, additional memory cell markers (e.g., IgG), CD19, CD38(plasma cell marker), and CD138 (plasma cell activation markers), CD154(B-cell activation marker), CTLA4 (negative B-cell co-stimulatormarker), CD45 (pan-leukocyte/lymphocyte marker), CD14, CD16, CD25, CD3,7-AAD, CD69, CD71, CD86 IFN gamma, IL-2, TNF alpha, CD45RA, CCR7 andCD54. Thus, in some embodiments, the method includes measuring thenumber of B cells that express one or more of CD27, IgM, IgA, IgG, IgD,CD5 and CD10. In other embodiments, the method includes measuring thenumber of memory B cells, plasma cells, or activated B cells. In someexamples, IgD+B-cells are considered to be naive B-cells. In otherexamples, a CD27+ or an IgG+ B-cell is considered to be a memory B-cell.In some examples, additional biomarkers include one or more B-cellmarkers disclosed in Linas et al. (Immunology Letters 134: 113-121,2011) which is hereby incorporated by reference in its entirety.

In some embodiments, expression levels of the plurality of biomarkersare measured using FACS. Any FACS technique (including variants based onprinciples of flow cytometry e.g., mass spectrometric visualization ofcellular markers with metallic ligands) or any other cellular imagingmay be employed as long as it is not detrimental to the viability of thedesired cells (for exemplary methods of FACS see U.S. Pat. No.5,061,620, herein incorporated by reference).

However, other techniques of differing efficacy may be employed toisolate and enumerate desired populations of cells. The separationtechniques employed should maximize the retention of viability of thefraction of the cells to be collected. The particular technique employedwill, of course, depend upon the efficiency of separation, cytotoxicityof the method, the ease and speed of separation, and what equipmentand/or technical skill is required. Separation procedures may includemagnetic separation, using antibody-coated paramagnetic beads, affinitychromatography, cytotoxic agents, either joined to a monoclonal antibodyor used in conjunction with complement, and “panning”, which utilizes amonoclonal antibody attached to a solid matrix, or another convenienttechnique. Antibodies attached to paramagnetic beads and other solidmatrices, such as agarose beads, polystyrene beads, hollow fibermembranes and plastic petri dishes, allow for direct separation. Cellsthat are bound by the antibody can be removed from the cell suspensionby simply physically separating the solid support from the cellsuspension. The exact conditions and duration of incubation of the cellswith the solid phase-linked antibodies will depend upon several factorsspecific to the system employed. The selection of appropriateconditions, however, is well within the skill in the art.

The unbound cells then can be eluted or washed away with physiologicbuffer after sufficient time has been allowed for the cells expressing amarker of interest (such as an antigen that binds one or more of themonoclonal antibodies disclosed herein) to bind to the solid-phaselinked antibodies. The bound cells are then separated from the solidphase by any appropriate method, depending mainly upon the nature of thesolid phase and the antibody employed.

In one example, the presence and quantity of the various biologicalmarkers are measured by labeling the cells with marker specific coloreddyes which are able to be detected and differentiated by a flowcytometer. Dyes known to those of ordinary skill in the art, such ascarboxy fluorescein diacetate succimidyl ester (CFSE, Molecular Probes,Eugen, Oreg.), EMA (cell viability dye), 7-AAD (cell viability dye) andQuantum dots, such as having emission spectra between 545 nm and 800 nm(Quantum Dot Corp. Hayward, Calif.), can be used to detect the desiredmarkers.

While any suitable equipment and methodology for measuring the multipleparameters can be employed, in one example a flow cytometer is used.Flow cytometers capable of detecting and differentiating at least 4 (andmore preferably at least 7) differently colored markers are employed. Insome examples, a flow cytometer capable of detecting and differentiatingat least 10, such as at least 15, at least 20, or at least 30 differentcolored markers is employed. In some examples, a flow cytometer capableof measuring and comparing in at least 25 or more multiple parameters,such as in excess of 50 multiple parameters or even over 100 multipleparameters is utilized. These flow cytometric capabilities exist innovel mass spectrometry platforms which detect metal dye labels. Methodsof using a flow cytometric machine are known to hose of skill in theart.

IV. Additional Methods

The disclosed methods can also be used to determine the B-cell responseto other antigens, including antigens from allergens, infectiouspathogens, tumors or associated with autoimmune diseases/disorders.Infectious pathogens include bacteria, fungi, protists, prions and/orviruses. These additional uses can be either alone or in addition todiagnosing or predicting organ transplant rejection.

In some embodiments, the method is used to detect reactivity to anantigen from a pathogen. For example, the method includes determining anAPI by comparing uptake of an antigen from a first pathogen to uptake ofan antigen from a second (reference) pathogen by APCs from a subject ofinterest. An API>1 indicates increased likelihood of infection with thefirst pathogen and a decreased likelihood of infection with the secondpathogen. For example, and API of greater than 1.2, greater than 1.5,greater than 1.75, greater than 2, greater than 3, greater than 4,greater than 5, greater than 6, greater than 7, greater than 8, greaterthan 9, or greater than 10 can indicate increased likelihood ofinfection with the first pathogen. An API<1, such as between 0.1 to 0.95or 0.3 to 0.85, indicates a decreased likelihood of infection with thefirst pathogen, and an increased likelihood of infection with the secondpathogen. For example, and API of less than 0.9, less than 0.75, lessthan 0.5, less than 0.1 or less than 0.1 can indicate a increasedlikelihood of an infection with the second pathogen, and a decreasedlikelihood of infection with the second pathogen.

In some embodiments of the method, determining the antigen presentingindex comprises contacting a first portion of the biological samplecomprising APCs obtained from a subject at risk of acquiring or known tohave a particular disease or condition, such as a viral, fungal orbacterial infection, with a first antigen from a first viral, fungal, orbacterial pathogen, under conditions sufficient to induce uptake of theantigen; contacting a second portion of the biological sample comprisingAPCs obtained from the subject at risk of acquiring or known to have aparticular disease or condition, with a second reference or anon-pathogenic antigen from different pathogen, under conditionssufficient to induce uptake of the second antigen; and determining theratio of uptake of the first antigen in the first portion of thebiological sample to uptake of the second antigen in the second portionof the biological sample. An increase in uptake of the first antigen ascompared to the uptake of the second (reference) antigen indicates thatthe subject has an infection with the first pathogen. An increase in theuptake of the second antigen as compared to the uptake of the firstantigen indicates that the subject has an infection with the secondpathogen. Another way in which the uptake of a pathogenic antigen canindicate disease severity is if it exceeds a threshold established inpatients with varying disease severity.

An API of greater than one indicates the presence of a particularcondition, such as an infection with the first pathogen, with asensitivity of at least 90% and indicates the presence of infection withspecificity of at least 90% for the risk of infection or acquiring thedisease or presence of infection or disease. In some examples, themethods disclosed herein have a sensitivity of at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98% or at least 99%. In some examples, themethods disclosed herein have a specificity of at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98% or at least 99%.

Similarly, an API of greater than one indicates the presence of aparticular condition, such as an infection with the first pathogen, witha sensitivity of at least 90% and a indicates the presence of infectionwith specificity of at least 90% for the risk of infection or acquiringthe disease or presence of infection or disease. In some examples, themethods disclosed herein have a sensitivity of at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98% or at least 99%. In some examples, themethods disclosed herein have a specificity of at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98% or at least 99%.

The first and second pathogen of interest can be any allergen, bacteria,fungus or virus including those described herein.

i. Viral Pathogens

Specific examples of viral pathogens include without limitation any oneor more of (or any combination of) Arenaviruses (such as Guanaritovirus, Lassa virus, Junin virus, Machupo virus and Sabia),Arteriviruses, Roniviruses, Astroviruses, Bunyaviruses (such asCrimean-Congo hemorrhagic fever virus and Hantavirus), Barnaviruses,Birnaviruses, Bornaviruses (such as Borna disease virus), Bromoviruses,Caliciviruses, Chrysoviruses, Coronaviruses (such as Coronavirus andSARS), Cystoviruses, Closteroviruses, Comoviruses, Dicistroviruses,Flaviruses (such as Yellow fever virus, West Nile virus, Hepatitis Cvirus, and Dengue fever virus), Filoviruses (such as Ebola virus andMarburg virus), Flexiviruses, Hepeviruses (such as Hepatitis E virus),human adenoviruses (such as human adenovirus A-F), human astroviruses,human BK polyomaviruses, human bocaviruses, human coronavirus (such as ahuman coronavirus HKU1, NL63, and OC43), human enteroviruses (such ashuman enterovirus A-D), human erythrovirus V9, human foamy viruses,human herpesviruses (such as human herpesvirus 1 (herpes simplex virustype 1), human herpesvirus 2 (herpes simplex virus type 2), humanherpesvirus 3 (Varicella zoster virus), human herpesvirus 4 type 1(Epstein-Barr virus type 1), human herpesvirus 4 type 2 (Epstein-Barrvirus type 2), human herpesvirus 5 strain AD169, human herpesvirus 5strain Merlin Strain, human herpesvirus 6A, human herpesvirus 6B, humanherpesvirus 7, human herpesvirus 8 type M, human herpesvirus 8 type Pand Human Cyotmegalovirus), human immunodeficiency viruses (HIV) (suchas HIV 1 and HIV 2), human metapneumoviruses, human papillomaviruses,human parainfluenza viruses (such as human parainfluenza virus 1-3),human parechoviruses, human parvoviruses (such as human parvovirus 4 andhuman parvovirus B19), human respiratory syncytial viruses, humanrhinoviruses (such as human rhinovirus A and human rhinovirus B), humanspumaretroviruses, human T-lymphotropic viruses (such as humanT-lymphotropic virus 1 and human T-lymphotropic virus 2), Human polyomaviruses, Hypoviruses, Leviviruses, Luteoviruses, Lymphocyticchoriomeningitis viruses (LCM), Marnaviruses, Narnaviruses, Nidovirales,Nodaviruses, Orthomyxoviruses (such as Influenza viruses),Partitiviruses, Paramyxoviruses (such as Measles virus and Mumps virus),Picornaviruses (such as Poliovirus, the common cold virus, and HepatitisA virus), Potyviruses, Poxviruses (such as Variola and Cowpox),Sequiviruses, Reoviruses (such as Rotavirus), Rhabdoviruses (such asRabies virus), Rhabdoviruses (such as Vesicular stomatitis virus,Tetraviruses, Togaviruses (such as Rubella virus and Ross River virus),Tombusviruses, Totiviruses, Tymoviruses, and Noroviruses among others.

Viral antigens may be from a Hepatitis C virus (HCV). HCV antigens maybe selected from one or more of E1, E2, E1/E2, NS345 polyprotein, NS345-core polyprotein, core, and/or peptides from the nonstructuralregions (Houghton et al. (1991) Hepatology 14:381-388, which isincorporated by reference).

Viral antigens may be derived from a Human Herpes virus, such as HerpesSimplex Virus (HSV), Varicella-zoster virus (VZV), Epstein-Barr virus(EBV), or Cytomegalovirus (CMV). Human Herpes virus antigens may beselected from immediate early proteins, early proteins, and lateproteins. HSV antigens may be derived from HSV-I or HSV-2 strains. HSVantigens may be selected from glycoproteins gB, gC, gD and gH, or immuneescape proteins (gC, gE, or gl). VZV antigens may be selected from core,nucleocapsid, tegument, or envelope proteins. A live attenuated VZVvaccine is commercially available. EBV antigens may be selected fromearly antigen (EA) proteins, viral capsid antigen (VCA), andglycoproteins of the membrane antigen (MA). CMV antigens may be selectedfrom capsid proteins, envelope glycoproteins (such as gB and gH), andtegument proteins. Exemplary herpes antigens include (GENBANK™ AccessionNo. in parentheses) those derived from human herpesvirus 1 (Herpessimplex virus type 1) (NC_001806), human herpesvirus 2 (Herpes simplexvirus type 2) (NC_001798), human herpesvirus 3 (Varicella zoster virus)(NC_001348), human herpesvirus 4 type 1 (Epstein-Barr virus type 1)(NC_007605), human herpesvirus 4 type 2 (Epstein-Barr virus type 2)(NC_009334), human herpesvirus 5 strain AD169 (NC_001347), humanherpesvirus 5 strain Merlin Strain (NC_006273), human herpesvirus 6A(NC_001664), human herpesvirus 6B (NC_000898), human herpesvirus 7(NC_001716), human herpesvirus 8 type M (NC_003409), and humanherpesvirus 8 type P (NC_009333).

Human Papilloma virus (HPV) antigens are known in the art and can befound for example in International Patent Publication No. WO96/19496,(incorporated by reference in its entirety) which discloses variants ofHPV E6 and E7 proteins, particularly fusion proteins of E6/E7 with adeletion in both the E6 and E7 proteins. HPV L1 based antigens aredisclosed in international Patent publication Nos. W094/00152,W094/20137, W093/02184 and W094/05792, all of which are incorporated byreference. Such an antigen can include the L1 antigen as a monomer, acapsomer or a virus like particle. Such particles may additionallycomprise L2 proteins. Other HPV antigens are the early proteins, such asE7 or fusion proteins such as L2-E7. Exemplary HPV antigens include(GENBANK™ Accession No. in parentheses) those derived from humanpapillomavirus-1 (NC_001356), human papillomavirus-18 (NC_001357), humanpapillomavirus-2 (NC_001352), human papillomavirus-54 (NC_001676), humanpapillomavirus-61 (NC_001694), human papillomavirus-cand90 (NC_004104),human papillomavirus RTRX7 (NC_004761), human papillomavirus type 10(NC_001576), human papillomavirus type 101 (NC_008189), humanpapillomavirus type 103 (NC_008188), human papillomavirus type 107(NC_009239), human papillomavirus type 16 (NC_001526), humanpapillomavirus type 24 (NC_001683), human papillomavirus type 26(NC_001583), human papillomavirus type 32 (NC_001586), humanpapillomavirus type 34 (NC_001587), human papillomavirus type 4(NC_001457), human papillomavirus type 41 (NC_001354), humanpapillomavirus type 48 (NC_001690), human papillomavirus type 49(NC_001591), human papillomavirus type 5 (NC_001531), humanpapillomavirus type 50 (NC_001691), human papillomavirus type 53(NC_001593), human papillomavirus type 60 (NC_001693), humanpapillomavirus type 63 (NC_001458), human papillomavirus type 6b(NC_001355), human papillomavirus type 7 (NC_001595), humanpapillomavirus type 71 (NC_002644), human papillomavirus type 9(NC_001596), human papillomavirus type 92 (NC_004500), and humanpapillomavirus type 96 (NC_005134).

Viral antigens may be derived from a Retrovirus, such as an Oncovirus, aLentivirus or a Spumavirus. Oncovirus antigens may be derived fromHTLV-I, HTLV-2 or HTLV-5. Lentivirus antigens may be derived from HIV-Ior HIV-2. Retrovirus antigens may be selected from gag, pol, env, tax,tat, rex, rev, nef, vif, vpu, and vpr. Antigens for HIV are known in theart, for example HIV antigens may be selected from gag (p24gag andp55gag), env (gp160 and gp41), pol, tat, nef, rev vpu, miniproteins,(p55 gag and gp140v). HIV antigens may be derived from one or more ofthe following strains: H1Vmb, HIV; HIVLAV, HIVLAI, HIVM N, HIV-1 CM235,HIV-1 US4. Examples of HIV antigens can be found in International PatentPublication Nos. WO09/089568, WO09/080719, WO08/099284, and WO00/15255,and U.S. Pat. Nos. 7,531,181 and 6,225,443, all of which areincorporated by reference. Exemplary HIV antigens include those derivedfrom human immunodeficiency virus 1 (NC_001802), human immunodeficiencyvirus 2 (NC_001722).

ii. Allergens

Exemplary allergens (which are nonparasitic antigens capable ofstimulating a type-I hypersensitivity reaction) include those derivedfrom plants, such as trees, for example Betula verrucosa allergens Bet v1, Bet v 2, and Bet v 4; Juniperous oxycedrus allergen Jun o 2; Castaneasativa allergen Cas s 2; and Hevea brasiliensis allergens Hev b 1, Hey b3, Hey b 8, Hey b 9, Hey b 10 and Hey b 11; grasses, such as Phleumpretense allergens Phl p 1, Phl p 2, Phl p 4, Phl p 5a, Phl p 5, Phl p6, Phl p 7, Phl p 11, and Phl p 12; weeds, such as Parietaria judaicaallergen Par j 2.01011; and Artemisia vulgaris allergens Art v 1 and Artv 3; Mites, such as Dermatophagoides pteronyssinus allergens Der p 1,Der p 2, Der p 5, Der p 7, Der p 8, and Der p 10; Tyrophaguputrescentiae allergen Tyr p 2; Lepidoglyphus destructor allergens Lep d2.01 and Lep d 13; and Euroglyphus maynei allergen Eur m 2.0101;animals, such as cats, for example Felis domesticus allergen Fel d 1;Penaeus aztecus allergen Pen a 1; Cyprinus carpo allergen Cyp c 1; andalbumin from cat, dog, cattle, mouse, rat, pig, sheep, chicken, rabbit,hamster, horse, pigeon, and guinea pig; Fungi, such as Penicilliumcitrinum allergens Pen c 3 and Pen c 19; Penicillium notatum allergenPen n 13; Aspergillus fumigatus allergens Asp f 1, Asp f3, Asp f 4, Aspf 6, Asp f 7 and Asp f 8; Alternaria alternata allergens Alt a 1 and Alta 5; Malassezia furfur allergen Mal f 1, Mal f 5, Mal f 6, Mal f 7, Malf 8, and Mal f 9; insects, such as Blatella germanica allergens Bla g 2,Bla g 4, and Bla g 5; Apis mellifera allergens Api m 2 and Api m 1;Vespula vulgaris allergen Ves v 5; Vespula germanica allergen Ves g 5;and Polstes annularis allergen Pol a 5; food, such as Malus domesticaallergens Mal d 1 and Mal d 2; Apium graveolens allergend Api g 1 andApi g 1.0201; Daucus carota allergen Dau c 1; and Arachis hypogaeaallergens Ara h 2 and Ara h 5 and the like.

iii. Bacterial Pathogen

Specific examples of bacterial pathogens include without limitation anyone or more of (or any combination of) Acinetobacter baumanii,Actinobacillus sp., Actinomycetes, Actinomyces sp. (such as Actinomycesisraelii and Actinomyces naeslundii), Aeromonas sp. (such as Aeromonashydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria), andAeromonas caviae), Anaplasma phagocytophilum, Alcaligenes xylosoxidans,Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillussp. (such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis,Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroidessp. (such as Bacteroides fragilis), Bartonella sp. (such as Bartonellabacilliformis and Bartonella henselae, Bifidobacterium sp., Bordetellasp. (such as Bordetella pertussis, Bordetella parapertussis, andBordetella bronchiseptica), Borrelia sp. (such as Borrelia recurrentis,and Borrelia burgdorferi), Brucella sp. (such as Brucella abortus,Brucella canis, Brucella melintensis and Brucella suis), Burkholderiasp. (such as Burkholderia pseudomallei and Burkholderia cepacia),Campylobacter sp. (such as Campylobacter jejuni, Campylobacter coli,Campylobacter lari and Campylobacter fetus), Capnocytophaga sp.,Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophilapneumoniae, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii,Corynebacterium sp. (such as, Corynebacterium diphtheriae,Corynebacterium jeikeum and Corynebacterium), Clostridium sp. (such asClostridium perfringens, Clostridium difficile, Clostridium botulinumand Clostridium tetani), Eikenella corrodens, Enterobacter sp. (such asEnterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacaeand Escherichia coli, including opportunistic Escherichia coli, such asenterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E.coli, enterohemorrhagic E. coli, enteroaggregative E. coli anduropathogenic E. coli) Enterococcus sp. (such as Enterococcus faecalisand Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensiaand Ehrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp.,Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis,Gemella morbillorum, Haemophilus sp. (such as Haemophilus influenzae,Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae,Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobactersp. (such as Helicobacter pylori, Helicobacter cinaedi and Helicobacterfennelliae), Kingella kingii, Klebsiella sp. (such as Klebsiellapneumoniae, Klebsiella granulomatis and Klebsiella oxytoca),Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans,Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp.,Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp.,Mycobacterium sp. (such as Mycobacterium leprae, Mycobacteriumtuberculosis, Mycobacterium intracellulare, Mycobacterium avium,Mycobacterium bovis, and Mycobacterium marinum), Mycoplasm sp. (such asMycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium),Nocardia sp. (such as Nocardia asteroides, Nocardia cyriacigeorgica andNocardia brasiliensis), Neisseria sp. (such as Neisseria gonorrhoeae andNeisseria meningitidis), Pasteurella multocida, Plesiomonasshigelloides. Prevotella sp., Porphyromonas sp., Prevotellamelaninogenica, Proteus sp. (such as Proteus vulgaris and Proteusmirabilis), Providencia sp. (such as Providencia alcalifaciens,Providencia rettgeri and Providencia stuartii), Pseudomonas aeruginosa,Propionibacterium acnes, Rhodococcus equi, Rickettsia sp. (such asRickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii,Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) andRickettsia typhi), Rhodococcus sp., Serratia marcescens,Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonellaenterica, Salmonella typhi, Salmonella paratyphi, Salmonellaenteritidis, Salmonella cholerasuis and Salmonella typhimurium),Serratia sp. (such as Serratia marcesans and Serratia liquifaciens),Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigellaboydii and Shigella sonnei), Staphylococcus sp. (such as Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus hemolyticus,Staphylococcus saprophyticus), Streptococcus sp. (such as Streptococcuspneumoniae (for example chloramphenicol-resistant serotype 4Streptococcus pneumoniae, spectinomycin-resistant serotype 6BStreptococcus pneumoniae, streptomycin-resistant serotype 9VStreptococcus pneumoniae, erythromycin-resistant serotype 14Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcuspneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae,tetracycline-resistant serotype 19F Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, andtrimethoprim-resistant serotype 23F Streptococcus pneumoniae,chloramphenicol-resistant serotype 4 Streptococcus pneumoniae,spectinomycin-resistant serotype 6B Streptococcus pneumoniae,streptomycin-resistant serotype 9V Streptococcus pneumoniae,optochin-resistant serotype 14 Streptococcus pneumoniae,rifampicin-resistant serotype 18C Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, ortrimethoprim-resistant serotype 23F Streptococcus pneumoniae),Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes,Group A streptococci, Streptococcus pyogenes, Group B streptococci,Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus,Streptococcus equismilis, Group D streptococci, Streptococcus bovis,Group F streptococci, and Streptococcus anginosus Group G streptococci),Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such asTreponema carateum, Treponema petenue, Treponema pallidum and Treponemaendemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonellasp., Vibrio sp. (such as Vibrio cholerae, Vibrio parahemolyticus, Vibriovulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrioalginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibriometchnikovii, Vibrio damsela and Vibrio furnisii), Yersinia sp. (such asYersinia enterocolitica, Yersinia pestis, and Yersiniapseudotuberculosis) and Xanthomonas maltophilia among others.

Bacterial antigens suitable for use in the disclosed methods includeproteins, polysaccharides, lipopolysaccharides, and outer membranevesicles which may be isolated, purified or derived from a bacterium. Inaddition, bacterial antigens include bacterial lysates and inactivatedbacteria formulations. Bacteria antigens can be produced by recombinantexpression. Bacterial antigens preferably include epitopes which areexposed on the surface of the bacteria during at least one stage of itslife cycle. Bacterial antigens include but are not limited to antigensderived from one or more of the bacteria set forth above as well as thespecific antigens examples identified below.

Neiserria gonorrhoeae antigens include Por (or porin) protein, such asPorB (see, e.g., Zhu et al. (2004) Vaccine 22:660-669), a transferringbinding protein, such as TbpA and TbpB (see, e.g., Price et al. (2004)Infect. Immun. 71(1):277-283), an opacity protein (such as Opa), areduction-modifiable protein (Rmp), and outer membrane vesicle (OMV)preparations (see, e.g., Plante et al. (2000) J. Infect. Dis.182:848-855); WO 99/24578; WO 99/36544; WO 99/57280; and WO 02/079243,all of which are incorporated by reference).

Chlamydia trachomatis antigens include antigens derived from serotypesA, B, Ba and C (agents of trachoma, a cause of blindness), serotypes L1,L3 (associated with Lymphogranuloma venereum), and serotypes, D-K.Chlamydia trachomas antigens also include antigens identified in WO00/37494; WO 03/049762; WO 03/068811; and WO 05/002619 (all of which areincorporated by reference), including PepA (CT045), LcrE (CT089), Art(CT381), DnaK (CT396), CT398, OmpH-like (CT242), L7/L12 (CT316), OmcA(CT444), AtosS (CT467), CT547, Eno (CT587), HrtA (CT823), MurG (CT761),CT396 and CT761, and specific combinations of these antigens.

Treponemapallidum (Syphilis) Antigens Unclude TmpA Antigen.

In some embodiments, a disclosed assay can be used to measure one ormore antigens derived from a sexually transmitted disease (STD). Suchantigens can provide for prophylactis or therapy for STDs such aschlamydia, genital herpes, hepatitis (such as HCV), genital warts,gonorrhea, syphilis and/or chancroid (see WO 00/15255, which isincorporated by reference). Antigens may be derived from one or moreviral or bacterial STDs. Viral STD antigens for use in the invention maybe derived from, for example, HIV, herpes simplex virus (HSV-I andHSV-2), human papillomavirus (HPV), and hepatitis (HCV). Bacterial STDantigens for use in the invention may be derived from, for example,Neiserria gonorrhoeae, Chlamydia trachomatis, Treponemapallidum,Haemophilus ducreyi, E. coli, and Streptococcus agalactiae.

iv. Fungal Pathogens

Exemplary fungal pathogens include one or more of Trichophyton rubrum,T. mentagrophytes, Epidermophyton floccosum, Microsporum canis,Pityrosporum orbiculare (Malassezia furfur), Candida sp. (such asCandida albicans), Aspergillus sp. (such as Aspergillus fumigatus,Aspergillus flavus and Aspergillus clavatus), Cryptococcus sp. (such asCryptococcus neoformans, Cryptococcus gattii, Cryptococcus laurentii andCryptococcus albidus), Histoplasma sp. (such as Histoplasma capsulatum),Pneumocystis sp. (such as Pneumocystis jirovecii), and Stachybotrys(such as Stachybotrys chartarum).

v. Parasites

Exemplary parasitic organisms include Malaria (Plasmodium falciparum, P.vivax, P. malariae), Schistosomes, Trypanosomes, Leishmania, Filarialnematodes, Trichomoniasis, Sarcosporidiasis, Taenia (T. saginata, T.solium), Leishmania, Toxoplasma gondii, Trichinelosis (Trichinellaspiralis) or Coccidiosis (Eimeria species).

vi. Tumor Antigens

Exemplary tumor antigens (antigens produced by tumor cells that canstimulate tumor-specific T-cell immune responses) include one or more ofthe following RAGE-1, tyrosinase, MAGE-1, MAGE-2, NY-ESO-1,Melan-A/MART-1, glycoprotein (gp) 75, gp100, beta-catenin,preferentially expressed antigen of melanoma (PRAME), MUM-1, Wilms tumor(WT)-1, carcinoembryonic antigen (CEA), and PR-1. Additional tumorantigens are known in the art (for example see Novellino et al., CancerImmunol. Immunother. 54(3):187-207, 2005) and are described below. Tumorantigens are also referred to as “cancer antigens.” The tumor antigencan be any tumor-associated antigen, which are well known in the art andinclude, for example, carcinoembryonic antigen (CEA), β-human chorionicgonadotropin, alphafetoprotein (AFP), lectin-reactive AFP,thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase,RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, macrophagecolony stimulating factor, prostase, prostate-specific antigen (PSA),PAP, NY-ESO-1, LAGE-1 a, p53, prostein, PSMA, Her2/neu, survivin andtelomerase, prostate-carcinoma tumor antigen-1, MAGE, ELF2M, neutrophilelastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-Ireceptor and mesothelin. A list of selected tumor antigens and theirassociated tumors are shown below.

Exemplary Tumors and their Tumor Antigens

Tumor Tumor Associated Target Antigens Acute myelogenous leukemia Wilmstumor 1 (WT1), PRAME, PR1, proteinase 3, elastase, cathepsin G Chronicmyelogenous leukemia WT1, PRAME, PR1, proteinase 3, elastase, cathepsinG Myelodysplastic syndrome WT1, PRAME, PR1, proteinase 3, elastase,cathepsin G Acute lymphoblastic leukemia PRAME Chronic lymphocyticleukemia Survivin Non-Hodgkin's lymphoma Survivin Multiple myelomaNY-ESO-1 Malignant melanoma MAGE, MART, Tyrosinase, PRAME GP100 Breastcancer WT1, Herceptin, epithelial tumor antigen (ETA) Lung cancer WT1Ovarian cancer CA-125 Prostate cancer PSA Pancreatic cancer CA19-9,RCAS1 Colon cancer CEA Cervical Cancer SCC, CA125, CEA, Cytokeratins(TPA, TPS, Cyfra21-1) Renal cell carcinoma (RCC) Fibroblast growthfactor 5 Germ cell tumors AFP

In some embodiments, the assay is to detect an infection with a pathogenin an immunocompromised subject. Immunocompromised subjects are moresusceptible to opportunistic infections, for example viral, fungal,protozoan, or bacterial infections, prion diseases, and certainneoplasms. Those who can be considered to be immunocompromised include,but are not limited to, subjects with AIDS (or HIV positive), subjectswith severe combined immune deficiency (SCID), diabetics, subjects whohave had transplants and who are taking immunosuppressives, and thosewho are receiving chemotherapy for cancer. Immunocompromised individualsalso includes subjects with most forms of cancer (other than skincancer), sickle cell anemia, cystic fibrosis, those who do not have aspleen, subjects with end stage kidney disease (dialysis), and those whohave been taking corticosteroids on a frequent basis by pill orinjection within the last year. Subjects with severe liver, lung, orheart disease also may be immunocompromised.

In other embodiments, the immunocompromised subject is infected with alentivirus. Lentiviruses include, but are not limited to humanimmunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type2 (HIV-2), simian immunodeficiency virus agm (SIVagm), simianimmunodeficiency virus mnd (SIVmnd), simian immunodeficiency virus syk(SIVsyk), simian immunodeficiency virus col (SIVcol), Visna-Maedi virus(VMV), bovine immunodeficiency virus (BIV), feline immunodeficiencyvirus (FIV), caprine arthritis-encephalitis virus (CAEV), and equineinfectious anemia virus (EIAV). In some embodiments, the lentivirus ishuman immunodeficiency virus type 1 (HIV-1). In some embodiments, thelentivirus is human immunodeficiency virus type 2 (HIV-2). It iscontemplated that pathogenic antigen can be an extract of the pathogen,or a synthetic protein or peptide fragment, or synthesized fragments ofan antigenic particle of the pathogen with overlapping amino acid ornucleotide sequence.

In some embodiments, the method further comprises detecting a specificset of immune cells, such as B cell, T cells or monocytes.

In one particular embodiment, the method includes assessing the uptakeor API of B-cells. In this example, API is positively correlated withCD154 expression of the T and B-cells stimulated with the hepatititsvirus or its fragments. An API of greater than 1 (hepatititsB/fragment/peptide uptake>uptake of reference antigen) indicates that asubject is at an increased risk of infection or the presence ofHepatitis B. Alternatively, if a threshold number of B-cells which takeup hepatitis B is exceeded, then the person is at increased risk ofhepatitis B.

In one particular embodiment, the method includes assessing the uptakeor API of monocytes. In this example, API is positively correlated withCD14 expression of monocytes stimulated with the hepatitis virus or itsfragments. An API of greater than 1 (hepatitits B/fragment/peptideuptake>uptake of reference antigen) indicates that a subject is at anincreased risk of infection or the presence of Hepatitis B.Alternatively, if a threshold number of monocytes which take uphepatitis B is exceeded, then the person is at increased risk ofhepatitis B.

In some examples, the plurality of biomarkers includes one or moremarkers for one or more of the pathogens listed above.

In further examples, a global assay is disclosed that allows diagnosingor predicting organ transplant rejection as described in detail inSection III in combination with diagnosing or predicting pathogeninfection, such as viral or bacterial infection. For example, specificmarkers known to those of skill in the art are utilized to detect aparticular viral and/or bacterial infection and at least one or more ofCD14, CD16, CD27, IgM, IgA, IgG, IgD, CD5 and CD10 markers are utilizedto diagnose or predict organ transplant rejection. It is contemplatedthat additional biomarkers can also be detected, such as additionalmemory cell markers (e.g., IgG), CD19, CD38 (plasma cell marker), andCD138 (plasma cell activation markers), CD154 (B-cell activationmarker), CTLA4 (negative B-cell co-stimulator marker) and CD45(pan-leukocyte/lymphocyte marker). In one example, any of the phenotypicmarkers disclosed herein are used alone or in combination.

In some embodiments, expression levels of the plurality of biomarkersare measured using flow cytometry or other methods known to those ofskill in the art, including those described herein (see Section III andExamples).

The disclosure is further illustrated by the following non-limitingExamples.

EXAMPLES Example 1 B-Cell Rejection Monitoring Assay

This example discloses a B-cell rejection monitoring assay foridentifying recipients at risk for ACR and HR.

In these data sets, the rejection-prone recipient was labeled “aRejector” and the rejection-free recipient “a Non-Rejector.”Significantly higher API distinguished without overlap (100% sensitivityand specificity) children who had biopsy-proven rejection (Rejectors)after liver (LTx) or small bowel transplantation (SBTx), from those whowere rejection-free (Non-Rejectors). (Table 1).

TABLE 1 LTx API (Median ± SEM) SBTx API (Median ± SEM) NR n = 20 0.750 ±0.048 NR n = 18 0.555 ± 0.060 R n = 15 1.794 ± 0.506 R n = 11 1.781 ±0.255 p-value 0.0030 p-value 0.0003

Additional data showed that various types of transplant recipientsshowed similar results. For example, similar results were observed inall B-cell compartments except the IgG+ compartment. These subsets,excluding IgG+ subsets are defined by the presence or absence of CD27,IgM, IgA, IgD, CD5, and CD10. The minimal numbers of markers includeCD19 or CD20 to label a cell as B-cell, IgG and CD27 to call it a memoryB-cell cell, and IgM or IgD to call it a naive B-cell. These studiesprovide support of the use of the API to diagnose ACR and HR and measureits risk.

API beared a significant positive correlation with inflammatorydonor-specific B-cells, which expressed the inflammatory marker CD154(allospecific CD154+TcM) (allospecific CD154+B-cells). Thesecorrelations were seen in either the liver or small bowel transplantrecipient populations (Tables 2 and 3). Similar results were observed inall B-cell compartments except the IgG+ compartment. These subsets,excluding IgG+ subsets were defined by the presence or absence of CD27,IgM, IgA, IgD, CD5, and CD10.

TABLE 2 Correlations for donor antigen uptake of B-cells (API) withCD154+ and CTLA-4+ T-cytotoxic memory & B-cells in Liver transplantrecipients. n = 14NR, 10R CD19+B-cells CD154+TcM (Spearman r) 0.5600 p:value 0.0044 CTLA4+TcM (Spearman r) −0.5316 p: value 0.0090 CD154+CD19(Spearman r) 0.6246 p: value 0.0019 CTLA4+CD19 (Spearman r) −0.6084 p:value 0.0027

TABLE 3 Correlations for donor antigen uptake of B-cells (API) withCD154+ and CTLA-4+ T-cytotoxic memory & B-cells in Small boweltransplant recipients. n = 17NR, 14R CD19+B-cells CD154+TcM (Spearman r)0.7855 p: value 0.00000017 CTLA4+TcM (Spearman r) −0.3931 p: value0.0349 CD154+CD19 (Spearman r) 0.4347 p: value 0.0145

API had a significant negative correlation with antiinflammatorydonor-specific T-cytotoxic memory cells which express theantiinflammatory marker CTLA4 (allospecific CTLA4+TcM). CTLA4+TcM weremeasured in a mixed leukocyte response (MLR). CTLA4+TcM could also bemeasured in the same study as the API, by extending the incubation from40 minutes to 6-8 hours. These correlations were seen in either theliver or small bowel transplant recipient populations (Tables 2 and 3).Additional data showed that all types of transplant recipients wouldshow similar results. Similar results were observed in all B-cellcompartments except the IgG+ compartment. These subsets, excluding IgG+subsets were defined by the presence or absence of CD27, IgM, IgA, IgD,CD5, and CD.

The IgG+ memory compartment of B-cells was less efficient at presentingdonor antigen in the presence of humoral rejection, which might occuralone, or with Acute cellular rejection. In contrast, the API of naiveB-cell compartments, such as IgD+ compartments, or the IgD+CD27−compartments continued to show API>1 when ACR is present without HR.

These observations can be used to distinguish between HR and ACR. Forexample, the API of IgG+B-cells (memory) is expressed a ratio with theAPI of naive IgD+B-cells. Effectively, this ratio is also a measure ofdonor antigen uptake by memory IgG+B-cells, relative to donor antigenuptake by the naive IgD+B-cells. The resulting memory:naive B-cell APIratio is <1 if humoral rejection is encountered alone or with ACR,and >1 if only ACR is present, without HR.

These studies support the use of the disclosed assay employing an API todiagnose ACR and HR as well as measure its risk.

Example 2 Organ Transplant Monitoring Assay

This example discloses an assay for identifying recipients at risk forACR and HR in which it provides the following indications: (1) analysisof B-cells which take up donor antigen; (2) characterization of theB-cell alloresponse (e.g., whether inflammatory or anti-inflammatory);and characterization of the T-cytotoxic memory cell alloresponse (e.g.,whether inflammatory or anti-inflammatory).

The API of B-cells, its naive and memory compartments, and the resultingproduction of CD154 and CTLA4 in T- and B-cells, which are described inExample 1 are all combined into a single 6-8 hour test, in whichdye-labeled donor and third-party antigen act as stimulators.Polychromatic flow cytometry is used to measure B-cell antigenpresentation, and B- and T-cell inflammatory or anti-inflammatoryalloresponse simultaneously. This combined assay provides acomprehensive analysis of B-cells which take up donor antigen, thecharacter of the B-cell alloresponse, whether inflammatory oranti-inflammatory, and the alloresponse of T-cytotoxic memory cells,whether inflammatory or anti-inflammatory.

Example 3 B-Cell Antigen Presentation Assay

The example provides a B-cell antigen-presentation assay for detectingrisk of rejection in subjects with a transplanted organ.

Assay System: Lymphocytes obtained from a transplant recipient weremixed with donor antigen or with third-party antigen. Third-partyantigen included antigen from an individual who is antigenicallydissimilar to the recipient or the donor. Antigenic similarity ordissimilarity was determined at the HLA loci. These histocompatibilityloci included the major class I (e.g., HLA-A, -B and -C) and class II(e.g. HLA-DR, -DP, -DQ, -DOA, -DOB and -DM) loci. If actual donorantigen was not available, antigen from normal human subjects which wasmatched with actual donor at the HLA loci was used. The ratio of donorantigen to third-party antigen uptake and presentation was the API. Ifthe donor antigen presentation exceeded that due to third-party, the APIwas usually >1 and the individual was at increased risk of rejection. Ifthe donor antigen presentation was exceeded by the third-party antigen,the API is usually <1 and the individual is at decreased risk ofrejection. As shown in FIG. 1, in the rejector (upper panels) 23.6%recipient B-cells presented donor antigen (middle upper panel), comparedwith 4.8% recipient B-cells which presented third-party antigen (rightupper panel) for an API of 4.91. In the non-rejector (lower panels),35.9% of recipient B-cells presented third-party antigen (lower rightpanel), but only 13.3% presented donor antigen (lower middle panel). TheAPI is 0.037 in this non-rejector.

The uptake and presentation of antigen was measured by placing donor orthird-party antigen in contact with either purified B-cells, orperipheral blood leukocytes (PBL) from the recipient. Thereafter,imaging techniques (such as flow cytometry, or a variety of microscopictechniques, e.g., confocal microscopy) were used to measure antigenwhich has been taken up by the B-cell.

Tables 4-7 summarize differences in the API between rejectors andnon-rejectors for some of the common subsets of B-cells in children whohad received liver (Table 4) or intestine transplantation (Table 5), orthe combined liver or intestine transplant population (Table 6) andadult renal transplant recipients (Table 7). The B-cell was identifiedwith the marker CD19. B-cell subsets were labeled with CD27, a memorymarker, and IgG+ another marker of B-cell memory. One measurement wasincluded per subject, made either in proximity to biopsy-provenrejection (rejector status) or an established rejection-free(non-rejector status). Within the liver or the intestine transplantcohort, some subjects had only received a single measurement, and othershad been monitored serially before transplantation, and at days 1-60 and61-200 after transplantation. The single measurement from seriallymonitored liver or intestine patients which has been included in Tables4-6 was one made during days 1-60 after transplantation.

TABLE 4 Differences in median ± SEM antigen presentation index (API)between non- rejectors and rejectors after liver transplantation.Outcome CD19+ CD19+CD27+ CD19+CD27− CD19+IgG+ CD19+IgG− Non-rejectors (n= 34) 0.512 ± 0.057 0.589 ± 0.083 0.433 ± 0.177 0.843 ± 0.108 0.738 ±0.305 Rejectors (n = 26) 1.794 ± 0.237 1.411 ± 0.243 1.738 ± 0.710 1.303± 0.151 1.703 ± 0.317 p-value 3.12E−07 0.0006 0.0015 0.011 0.005(Legend: B-cell = CD19+, Memory B-cell = CD19+CD27+, naïve B-cell =CD19+CD27−, Memory IgG+B-cell = CD19+IgG+, Naïve IgG-B-cell =CD19+IgG−.).

TABLE 5 Differences in median ± SEM antigen presentation index (API)between non- rejectors and rejectors after intestine transplantation.Outcome CD19+ CD19+CD27+ CD19+CD27− CD19+IgG+ CD19+IgG− Non-rejectors (n= 34) 0.601 ± 0.048 0.660 ± 0.072 0.546 ± 0.097 0.620 ± 0.133 0.553 ±0.067 Rejectors (n = 22)  1.94 ± 0.427 1.386 ± 0.218 2.586 ± 0.957 0.968± 0.396 2.333 ± 0.980 p-value 4.38E−05 0.0001 0.001 0.042 0.014 (Legend:see Table 4).

TABLE 6 Differences in median ± SEM antigen presentation index (API)between non- rejectors and rejectors in the combined population of liveror intestine transplant recipients shown in Tables 4 and 5. CombinedCD19+ CD19+CD27+ CD19+CD27− CD19+IgG+ CD19+IgG− Non-rejectors (n = 68)0.543 ± 0.037 0.625 ± 0.054 0.477 ± 0.100 0.677 ± 0.088 0.608 ± 0.141Rejectors (n = 48)  1.79 ± 0.235 1.411 ± 0.164 2.058 ± 0.578 1.208 ±0.197 1.898 ± 0.470 p-value 2.86E−10 2.21E−07 4.1E−06 0.002 0.0002(Legend: see Table 4).

TABLE 7 Differences in median ± SEM antigen presentation index (API) ofCD19+B-cells between non-rejectors and rejectors after renaltransplantation. Combined CD19+API Non-rejectors (n = 10) 0.624 ± 0.14Rejectors (n = 7) 1.49 ± 3.6 p-value (one tail) 0.039

Assay performance was then evaluated with sensitivity and specificitytesting using the illustrative dataset summarized in Tables 4-6 forchildren with liver (Table 4) or intestine transplantation (Table 5) orthe combined cohort of liver or intestinal transplant recipients (Table6). The sensitivity was the proportion of rejectors with an APIexceeding the rejection-risk threshold. The specificity was theproportion of non-rejectors with an API below the rejection-riskthreshold.

Rejection-risk thresholds were identified and tested for each subjectpopulation using logistic regression and screening-replication testing.Liver recipients shown in Table 4 were divided into a screening cohortof 43 recipients in whom a single (cross-sectional) API measurement wasavailable in proximity to biopsy-proven rejection or an establishednon-rejector course. The sensitivity and specificity of this thresholdwas then confirmed in 17 remaining liver recipients called a replicationcohort, in whom API measurements were made longitudinally, beforetransplantation and at 1-60 and 61-200 days after liver transplantation.For replication purposes, the sensitivity and specificity of therejection-risk threshold identified in the screening cohort werere-tested using pre-transplant and 1-60-day API data.

In a manner similar to liver recipients, intestine transplant patientssummarized in Table 5 consisted of 45 cross-sectionally monitoredrecipients who made up the screening cohort, and the remaining 11longitudinally monitored recipients, who made up the replication cohort.

Table 8 below shows the rejection-risk thresholds for B-cell antigenpresentation (CD19+cells) derived from liver recipients, intestinerecipients, and the combined population which make up the respectivescreening (cross-sectional) cohorts. The thresholds at or above whichrejector status was predicted for the liver, intestine or the combinedscreening cohorts were 1.115, 1.115 and 1.108, respectively. Thesensitivity and specificity of these thresholds were confirmed in therespective replication cohorts, for two of three longitudinal APImeasurements, the pre-transplant API and the post-transplant API at 1-60days. For each sensitivity value, the numbers of rejectors identifiedcorrectly, for e.g. 11 of 13 liver recipients with API≥1.115 are shownin the Table. Similarly, the numbers of non-rejectors identified ashaving API below the rejection-risk threshold are also shown.

TABLE 8 Summary of sensitivity and specificity testing of therejection-risk threshold API in children with liver or intestinetransplantation or the combined population Screening cohort thresholdAPI Sensitivity Specificity Cross sectional API Liver ≥1.115 84.6% (11of 13) 96.7% (29 of 30) Intestine ≥1.115 100% (15 of 15) 96.7% (29 of30) Combined ≥1.108 92.9% (26 of 28) 96.7% (58 of 60) Replication cohortthreshold API Sensitivity Specificity Pre-transplant API Liver ≥1.11587.5% (7 of 8) 100% (2 of 2) Intestine ≥1.115 100% (3 of 3) 100% (3 of3) Combined ≥1.108 90.9% (10 of 11) 100% (5 of 5) Post-transplant API atLiver ≥1.115 100% (13 of 13) 100% (4 of 4) days 1-60 Intestine ≥1.115100% ((7 of 7) 100% (4 of 4) Combined ≥1.108 100% (20 of 20) 100% (8 of8)

The dynamic nature of the API in the same individual was illustrated for28 children who had been monitored serially, before transplantation andat days 1-60 and 61-200 after liver or intestine transplantation (FIG.2). These results show that rejectors (who experience rejection withinthe first 60 days after transplantation) show increased risk ofrejection in the form of an API at or above the rejection-risk thresholdAPI 1.11 before transplantation. Rejectors also shows increased risk ofrejection during the rejection-prone period of 1-60 days, but showreduced rejection risk characterized by an API<1.11 during the laterpart of the follow-up. In contrast, most non-rejectors are likely toshow reduced risk of rejection characterized by an API<1.11 beforetransplantation which is likely to persist through the post-transplantcourse.

A benefit of the B-cell antigen presenting assay is its ability toutilize a “surrogate donor” antigen instead of actual donor antigen.Actual donor antigen usually consists of peripheral blood leukocytes orspleen cells called splenocytes which are obtained from the donor andconsumed during the tissue typing tests required at each of many centersthat receive the various organs from a donor for transplantation.Lifelong testing using actual donor cells is therefore not possible. Anillustrative study summarized in FIG. 3 shows that whether actual donorantigen was used, or surrogate donor antigen is used, the assignment ofrejector or rejector status does not change if a rejection-riskthreshold of 1.115 is used. Four non-rejectors and 2 rejectors have beentested simultaneously using actual donor and surrogate donor stimulatorsin this study. For each recipient, the same third-party stimulator wasused to calculate the API with either actual donor or surrogate donorstimulator.

Example 4 Antigen Presentation Assay with Monocytes

The example indicates the disclosed antigen-presentation assay isapplicable to additional types of antigen presenting cells such asmonocyte and dendritic cells.

Monocytes present antigen, and are precursors of monocyte-deriveddendritic cells. Therefore, monocyte function is representative offunction of monocyte-derived dendritic cells.

Presentation of CFSE-labeled antigenic lysate from donor or third-party,by CD14+monocytes, was evaluated in single samples from 26 recipientsand serial samples from 9 recipients of the liver or intestine. Anantigen presenting index was calculated as the ratio of donor antigenpresented by monocytes to third-party antigen presented by monocytes.

In 26 recipients with either liver (LTx) or intestine (ITx)transplantation, who were sampled once, cross-sectionally, during astable rejection-free period (non-rejectors, NR) or in proximity tobiopsy-proven rejection (rejectors, R), higher donor antigenpresentation relative to third-party antigen presentation was present,and led to a significantly greater API among rejectors compared withnon-rejectors (p=0.012, FIG. 4, Table 9). This cross-sectional cohortconsisted of 17 liver recipients (11 non-rejectors and 6 rejectors), and9 intestine transplant recipients (6 non-rejectors and 3 rejectors).

TABLE 9 Monocyte API in cross-sectional cohort (LTx = 11NR. 6R: ITx =6NR, 3R) mean ± SEM NR (17) 0.52 ± 0.098 R (9) 2.74 ± 0.684 p: value0.012

Nine children were sampled at a minimum of two of threetimepoints-before transplantation, and during 1-60 days and 61-200 days.Monocyte API was numerically higher among rejectors (FIG. 5, Table 9).When data from the 1-60-day rejection-prone period in this cohort waspooled with that from the cross-sectional cohort, significantly higherAPI was again seen in 16 total rejectors, compared with 19 totalnon-rejectors (Table 10, FIG. 6).

TABLE 10 Monocyte API-Longitudinal cohort (2NR, 7R) Pre-Tx 1-60 days61-200 days n = 2NR, 3R n = 2NR. 7R n = 2NR, 6R NR 0.71 ± 0.23 0.99 ±0.55 0.62 ± 0.01 R 3.5 ± 1.11 2.17 ± 0.45 2.92 ± 0.91 p: value 0.06 0.090.05 (one tail)

TABLE 11 Combined data for monocyte API from cross-sectional cohort andfrom the 1-60 day period in the longitudinal cohort in table form. LTxITx Combined mean ± SEM (n = 13NR, 11R) (n = 6NR, 5R) (n = 19NR, 16R) NR0.52 ± 0.13 0.66 ± 0.17 0.56 ± 0.1  R 2.58 ± 0.55 2.29 ± 0.69 2.49 ±0.42 p: value 0.0039 0.07 0.0004 W

Single measurements from cross-sectional cohort were combined with thosefrom the 1-60 day time period in the longitudinal cohort. Themeasurements were made in 16 rejectors and 19 non-rejectors. Fromlogistic regression analysis, a threshold API of 1.2 was obtained, at orwhich rejector status was predicted with sensitivity of 75% (12 of 16with API≥1.2), and specificity of 95% (18 of 19 with API<1.2). Rejectorswere sampled at mean interval of 3.6 days before initiating treatment ofbiopsy-proven rejection. One of 16 rejectors was samples 42 days afterthe rejection event.

The present studies demonstrated that B-cell antigen presentationmirrored antigen presentation by other types of antigen presenting cellssuch as the monocyte. A significant correlation (Spearman r=0.839,p=2.8E-10) was found between the API of B-cells and the API of monocytesin the 35 samples from which abovementioned data is derived (FIG. 7).These studies further indicate that monocyte antigen presentationpredicts rejection and non-rejector outcomes with high sensitivity andspecificity. Indices for presentation of antigen by monocytes andB-cells are significantly correlated. Therefore, the presently disclosedassay is not only applicable to B-cell antigen presentation, but toantigen presentation by other types of antigen presenting cells such asthe monocyte, and dendritic cells.

While this disclosure has been described with an emphasis uponparticular embodiments, it will be obvious to those of ordinary skill inthe art that variations of the particular embodiments may be used, andit is intended that the disclosure may be practiced otherwise than asspecifically described herein. Features, characteristics, compounds, orexamples described in conjunction with a particular aspect, embodiment,or example of the invention are to be understood to be applicable to anyother aspect, embodiment, or example of the invention. Accordingly, thisdisclosure includes all modifications encompassed within the spirit andscope of the disclosure as defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A method, comprising: (i) contacting a first portion of a biological sample comprising antigen presenting cells (APCs) obtained from a subject in need of or having received an organ transplant from a donor, with a donor antigen from the donor in vitro under conditions sufficient to induce uptake of the donor antigen; (ii) contacting a second portion of the biological sample comprising APCs obtained from the subject in need of or having received an organ transplant with a third-party antigen in vitro under conditions sufficient to induce uptake of the third-party antigen; and (iii) measuring amount of uptake of the donor antigen in the first portion of the biological sample and measuring amount of uptake of the third-party antigen in the second portion of the biological sample, wherein the APCs are dendritic cells or macrophages, wherein the method determines the ratio of uptake of the donor antigen in the first sample and the uptake of the third party antigen by the APCs in the second sample.
 2. The method of claim 1, wherein measuring amount of uptake of a donor antigen to uptake of a third-party antigen comprises (i) detecting at least one biomarker following treatment with the donor antigen with the first portion of the biological sample and the third-party antigen with the second portion of the biological sample; and (ii) measuring the amount of the at least one biomarker detected following treatment with donor antigen to the amount of the at least one biomarker detected following treatment with the third-party antigen to determine an antigen presenting index.
 3. The method of claim 1, wherein measuring the amount of uptake of the donor antigen to the amount of uptake of third-party antigen further comprises labeling the donor antigen and third-party antigen with a fluorescent molecule prior to contacting the first portion of the biological sample with the donor antigen and the second portion of the biological sample with the third-party antigen.
 4. The method of claim 1, wherein the method is used to diagnose or predict organ transplant rejection, diagnose or predict graft versus host disease or predict immunity.
 5. The method of claim 1, wherein the method is used to diagnose or predict graft versus host disease after solid organ, bone-marrow, or stem cell transplantation, or a combination thereof.
 6. The method of claim 1, wherein the method is used to predict immunity to an autoantigen, tumor antigen, pathogen antigen, or a combination thereof.
 7. The method of claim 1, wherein the APCs are macrophages.
 8. The method of claim 1, wherein the subject is a solid organ, bone marrow, stern cell, or combination thereof, recipient or candidate recipient.
 9. The method of claim 1, wherein the subject is a child.
 10. The method of claim 1, wherein the subject has acute cellular and/or humoral rejection.
 11. The method of claim 1, wherein the organ is a liver.
 12. The method of claim 1, wherein the organ is intestine.
 13. The method of claim 1, wherein the subject is receiving an immunosuppressive regimen.
 14. The method of claim 1, wherein the APCs are dendritic cells.
 15. The method of claim 1, wherein the donor antigen, the third-party antigen, or both, are labeled with a detectable label.
 16. The method of claim 15, wherein the label is a fluorochrome or a metallic ligand. 